JP2023052910A - Compound or salt thereof having affinity substance for soluble protein, cleavable moiety, and reactive group - Google Patents

Abstract

PROBLEM TO BE SOLVED: To develop a technique that allows modification of soluble proteins, in particular, regioselective modification of soluble proteins.

SOLUTION: The present invention provides a compound or salt thereof having an affinity substance for soluble proteins, a cleavable moiety, and a reactive group, represented by the following formula (I): A-L-B-R (where A is an affinity substance for soluble proteins, L is a cleavable linker that is a divalent group containing a cleavable moiety, (B) is (a) a divalent group containing a bioorthogonal functional group or (b) a divalent group not containing a bioorthogonal functional group, and R is a reactive group with the soluble proteins).

SELECTED DRAWING: None

COPYRIGHT: (C)2023,JPO&INPIT

Description

The present invention relates to compounds having an affinity for soluble proteins, cleavable moieties and reactive groups, salts thereof, and the like.

In recent years, research and development of antibody-drug conjugation (ADC) have been actively carried out. ADC, as its name suggests, is a drug in which a drug (eg, an anticancer drug) is conjugated to an antibody, and has direct cell-killing activity against cancer cells and the like. A representative ADC is T-DM1 (trade name: Kadcyla (registered trademark)) jointly developed by Immunogen and Roche (Non-Patent Documents 1 to 3).

ADCs including the T-DM1 have had a problem of non-uniformity since the beginning of their development. That is, since low-molecular-weight drugs are randomly reacted with about 70 to 80 Lys residues in an antibody, the drug-antibody ratio (DAR) and conjugation positions are not constant. It has been found that such random conjugation methods usually result in DARs in the range of 0 to 8, resulting in multiple drugs with different binding numbers of drugs. In recent years, it has been reported that changing the binding number and binding position of a drug to an ADC changes its pharmacokinetics, drug release rate, and effect. For these reasons, next-generation ADCs are required to control the number and positions of drugs to be conjugated. If the number and position are constant, it is believed that expected efficiency, variations in conjugation drugs, and lot differences, so-called regulation problems, can be resolved (Non-Patent Document 4).

Site-selective modification methods of antibodies have been studied all over the world, but most of them are modification methods using genetic engineering techniques or enzymes. With respect to genetic engineering modification methods, although site selectivity and number selectivity can be controlled, problems such as a decrease in the expression efficiency of the antibody itself (a decrease in the overall yield when preparing ADC) have been pointed out. In addition, there is a problem that it takes a long time to construct an antibody expression system (Non-Patent Documents 5 to 7).

In recent years, a method for chemically modifying proteins using small molecule probes in a crowded environment such as inside a cell has also been reported. This method is used for identification of receptors for imaging and repositioning of low-molecular-weight drugs. In addition, in the field of chemical biology, organic chemical protein modification methods using synthetic small molecule probes have attracted attention (Non-Patent Documents 8 to 10).

Recently, the C-CAP (Chemical Conjugation by Affinity Peptide) method has been developed. The method uses affinity peptides (Affinity
Succeeded in regioselective modification of antibodies by reacting peptide reagents with NHS-activated esters and drugs linked to Peptide) with antibodies (i.e., creating ADCs via linkers containing peptide moieties). are doing. This method was the first in the world to succeed in regioselectively modifying an antibody Fc region with a drug by a chemical synthesis technique, and the results were also good in practical use [reaction time: 30 minutes, yield: 70%. (for DAR 1), 100% regioselectivity] is confirmed. It has been demonstrated that the DAR can be controlled at 2 by adding about 5 equivalents of a peptide reagent, which is epoch-making in that the modification position can also be controlled (Patent Document 1).

WO2016/186206

Reichert JM et al. , Nat Biotechnol 2005;23:1073-8. Kubota T et al. , Cancer Sci 2009;100:1566-72. Wu AM et al. , Nat Biotechnol 2005;23:1137-46. Junutula JR et al. , Nat Biotechnol 2008;26:925-32. Shen BQ et al. , Nat Biotechnol 2012;30:184-9. Hofer T et al. , Biochemistry 2009;48:12047-57 Liu W et al. , Nat Methods 2007;4:239-44. S. T. Laughlin et al. , Science 2008; 320, 664 A. E. Speers et al. , ChemBioChem 2004; 5, 41 Y. Takaoka et al. , Angew. Chem. Int. Ed. 2013; 52,4088

An object of the present invention is to develop a technique that enables modification of soluble proteins, in particular, site-selective modification of soluble proteins.

As a result of intensive studies, the present inventors found that (1) an affinity substance for a soluble protein, (2) a reactive group for an amino acid residue that constitutes the soluble protein, and (3) the affinity substance and the reaction (4) a structural unit having a bioorthogonal functional group on the reactive group side by cleavage at the cleavable moiety (i.e., containing a bioorthogonal functional group and a reactive group) We have found that compounds developed based on a novel and original design concept that have structural features that can generate a structural unit) are useful for site-specific modification of soluble proteins (e.g., Fig. 1- 1, Fig. 1-2, Fig. 1-3, Fig. 2). The present inventors have also found that by using such compounds, soluble proteins (e.g., antibody-drug conjugates (ADC)) regioselectively have functional substances (e.g., drugs) that do not contain peptide moieties as linkers. ) can be prepared. Avoidance of the use of linkers containing peptide moieties that are potentially immunogenic and susceptible to hydrolysis in blood is desirable in clinical applications of ADCs. In other words, the method developed by the present inventors was the first in the world to succeed in regioselectively modifying an antibody Fc region with a drug by a chemical synthesis technique and without using a linker containing a peptide moiety. be able to. The present inventors have also succeeded in developing various compounds having the structural characteristics of (1) to (4) above (e.g., FIGS. 1-1, 1-2, 1-3 , FIG. 2), and completed the present invention.

That is, the present invention is as follows.

〔ここで、結合に直交する波線は、切断部位を示し、
複数のＲ_２ａ、複数のＲ_２ｂ、および複数のＲ_２ｃは、同一または異なって、
（ｉ）水素原子、またはハロゲン原子；
（ｉｉ）１価の炭化水素基；
（ｉｉｉ）アラルキル；
（ｉｖ）１価の複素環基；
（ｖ）Ｒ_ｃ－Ｏ－、Ｒ_ｃ－Ｃ（＝Ｏ）－、Ｒ_ｃ－Ｏ－Ｃ（＝Ｏ）－、もしくはＲ_ｃ－Ｃ（＝Ｏ）－Ｏ－（Ｒ_ｃは、水素原子、もしくは一価の炭化水素基を示す。）；
（ｖｉ）ＮＲ_ｄＲ_ｅ－、ＮＲ_ｄＲ_ｅ－Ｃ（＝Ｏ）－、ＮＲ_ｄＲ_ｅ－Ｃ（＝Ｏ）－Ｏ－、もしくはＲ_ｄ－Ｃ（＝Ｏ）－ＮＲ_ｅ－（Ｒ_ｄおよびＲ_ｅは、同一もしくは異なって、水素原子、もしくは一価の炭化水素基を示す。）；または
（ｖｉｉ）ニトロ基、硫酸基、スルホン酸基、シアノ基、もしくはカルボキシル基
からなる群より選ばれ、
Ｊは、－ＣＨ_２－、－Ｏ－、または－Ｓ－であり、
ｒは、１～４の任意の整数であり、
○（白丸）はＡに対する結合を示し、●（黒丸）はＢに対する結合を示す。
化学構造が、切断部位を中心にして非対称である場合、●がＡに対する結合を示し、○がＢに対する結合を示していてもよい。〕からなる群より選ばれるいずれか一つの化学構造に対応する、〔１〕～〔２０〕のいずれかの化合物またはその塩。
〔２４〕前記（ｉ）の切断性部分が、以下：

〔ここで、結合に直交する波線は、切断部位を示し、
Ｒ_２ａは、〔２３〕と同じであり、
○（白丸）はＡに対する結合を示し、●（黒丸）はＢに対する結合を示す。
化学構造が、切断部位を中心にして非対称である場合、●がＡに対する結合を示し、○がＢに対する結合を示していてもよい。〕からなる群より選ばれるいずれか一つの化学構造に対応する、〔２〕～〔１９〕、〔２１〕、〔２３〕のいずれかの化合物またはその塩。
〔２５〕前記（ｉｉ）の切断性部分が、以下：

〔ここで、結合に直交する波線は、切断部位を示し、
Ｒ_２ｂ、Ｒ_２ｃ、Ｊ、ｒは、〔２３〕と同じであり、
○（白丸）はＡに対する結合を示し、●（黒丸）はＢに対する結合を示す。
化学構造が、切断部位を中心にして非対称である場合、●がＡに対する結合を示し、○がＢに対する結合を示していてもよい。〕からなる群より選ばれるいずれか一つの化学構造に対応する、〔２〕～〔１９〕、〔２２〕、〔２３〕のいずれかの化合物またはその塩。
〔２６〕Ｌが、下記式（Ｌ１）～（Ｌ３）：
Ｌａ－Ｃ－Ｌｂ （Ｌ１）
Ｌａ－Ｃ （Ｌ２）
Ｃ－Ｌｂ （Ｌ３）
〔式中、
ＬａおよびＬｂは、それぞれ、２価の基であり、
Ｃは、切断性部分である。〕のいずれか一つで表される、〔１〕～〔２５〕のいずれかの化合物またはその塩。
〔２７〕前記ＬａおよびＬｂが、それぞれ、下記（Ｌａ’）および（Ｌｂ’）：

〔式中、
ｐおよびｐ’は、同一または異なって、０～１０の任意の整数であり、
ｑおよびｑ’は、同一または異なって、０～１０の任意の整数であり、
ＸおよびＸ’は、同一または異なって、炭素原子、窒素原子、または単結合（ここで、Ｘが窒素原子である場合、Ｒ_１ｂは存在せず、Ｘ’が窒素原子である場合、Ｒ_１ｂ’は存在しない。Ｘが単結合である場合、Ｒ_１ａおよびＲ_１ｂは存在せず、Ｘ’が単結合である場合、Ｒ_１ａ’およびＲ_１ｂ’は存在しない）であり、
Ｒ_１ａ、Ｒ_１ｂ、Ｒ_１ａ’およびＲ_１ｂ’は、同一または異なって、前記（ｉ）～（ｖｉｉ）からなる群より選ばれる原子または基である。〕で表される、〔２６〕の化合物またはその塩。
〔２８〕生体直交性官能基を含む２価の基が、アジド残基、アルデヒド残基、チオール残基、アルキン残基、アルケン残基、テトラジン残基、ニトロン残基、ヒドロキシルアミン残基、ニトリル残基、ヒドラジン残基、ケトン残基、ボロン酸残基、シアノベンゾチアゾール残基、アリル残基、ホスフィン残基、マレイミド残基、ジスルフィド残基、チオエステル基、α―ハロカルボニル残基、イソニトリル残基、シドノン残基、セレン残基からなる群より選ばれる生体直交性官能基を主鎖に含む２価の基である、〔１〕～〔２７〕のいずれかの化合物またはその塩。
〔２９〕生体直交性官能基を含む２価の基が、アジド残基、アルデヒド残基、チオール残基、アルキン残基、アルケン残基、ハロゲン残基、テトラジン残基、ニトロン残基、ヒドロキシルアミン残基、ニトリル残基、ヒドラジン残基、ケトン残基、ボロン酸残基、シアノベンゾチアゾール残基、アリル残基、ホスフィン残基、マレイミド残基、ジスルフィド残基、α―ハロカルボニル残基、イソニトリル残基、シドノン残基、セレン残基からなる群より選ばれる生体直交性官能基を側鎖に含む２価の基である、〔１〕～〔２７〕のいずれかの化合物またはその塩。
〔３０〕生体直交性官能基が、下記：

〔式中、
Ｒ_１ｆ、単一もしくは複数のＲ_１ｇおよび単一もしくは複数のＲ_１ｈは、同一もしくは異なって、前記（ｉ）～（ｖｉｉ）からなる群より選ばれる原子もしくは基、または電子吸引基であり、
・は、結合手である。）で表されるいずれか一つである、〔１〕～〔２９〕のいずれかの化合物またはその塩。
〔３１〕前記（ｂ）の２価の基が、置換されていてもよいアルキレン、置換されていてもよいシクロアルキレン、置換されていてもよいアリール、置換されていてもよい２価の複素環基、－ＮＲ_ａ－（Ｒ_ａは水素原子、または置換基を示す）、－Ｏ－、またはこれらの２以上の組み合わせからなる群より選ばれる、〔１〕～〔３０〕のいずれかの化合物またはその塩。
〔３２〕Ｂが、下記式（Ｂ－１）：

〔式中、
Ｙは、－ＮＨ－、－Ｏ－、－ＣＨ_２－、または下記式（Ｂ－２）：

（式中、
ＶおよびＶ’は、同一または異なって、－ＮＨ－、－Ｏ－、－ＣＨ_２－、または単結合であり、
Ｖ１は、生体直交性官能基を含む２価の基であり、
ｓは、０～１０の任意の整数であり、
式（Ｂ－２）における○および●は、それぞれ、式（Ｂ－１）における○および●と同じ配向である。）であり、
Ｚは、酸素原子、硫黄原子、または水素原子（Ｚが水素原子である場合、－Ｃ（＝Ｚ）－は、－ＣＨ_２－を示す。）であり、
式（Ｂ－１）における○（白丸）は、Ｌ側の部分に対する結合を示し、●（黒丸）はＲ側の部分に対する結合を示す。〕で表される、〔１〕～〔３１〕のいずれかの化合物またはその塩。
〔３３〕反応性基が、リジン残基、チロシン残基、またはトリプトファン残基のいずれか一つの側鎖に対して特異的な反応性基である、〔１〕～〔３２〕のいずれかの化合物またはその塩。
〔３４〕反応性基が、リジン残基の側鎖に対して特異的な反応性基である、〔３３〕の化合物またはその塩。
〔３５〕反応性基が、下記：

〔ここで、
Ｒ_５ａおよびＲ_５cは、前記（ｉ）～（ｖｉｉ）からなる群より選ばれる原子または基
であり、
Ｒ_５ｂは、電子吸引基であり、
ｊは、１～５の任意の整数であり、
ｋは、１～４の任意の整数である。〕からなる群より選ばれるいずれか一つの化学構造に対応する、〔１〕～〔３４〕のいずれかの化合物またはその塩。
〔３６〕ＡおよびＲを連結する主鎖の原子数が４～２０個である、〔１〕～〔３５〕のいずれかの化合物またはその塩。
〔３７〕ＡおよびＲを連結する主鎖が環構造を含まない、〔１〕～〔３６〕のいずれかの化合物またはその塩。
〔３８〕Ｌ－Ｂで表される部分構造がペプチド部分を含まない、〔１〕～〔３７〕のいずれかの化合物またはその塩。
〔３９〕前記式（Ｉ）で表される化合物が、下記（Ｉ’）：
Ａ－Ｂ２－Ｌ’－Ｂ１－Ｒ （Ｉ’）
〔式中、
ＡおよびＲは、前記式（Ｉ）のものと同じであり
Ｌ’は、切断性部分を含む２価の基である切断性リンカーであり、
Ｂ１およびＢ２は、同一または異なって、（ａ）生体直交性官能基を含む２価の基、または（ｂ）生体直交性官能基を含まない２価の基であり、
Ｂ１およびＢ２は、Ｌ’を中心にした対称構造を有していてもよい。〕で表される化合物である、〔１〕～〔３８〕のいずれかの化合物またはその塩。
〔４０〕前記式（Ｉ’）で表される化合物が、下記（Ｉ’’）：

〔式中、
ＡおよびＲは、〔１〕記載の式（Ｉ）のものと同じであり、
Ｃは、切断性部分であり、
ｐ、ｐ’、ｑ、ｑ’、Ｘ、Ｘ’、Ｒ_１ａ、Ｒ_１ａ’、Ｒ_１ｂ、およびＲ_１ｂ’は、〔２７〕記載の式（Ｌａ’）および（Ｌｂ’）のものと同じであり、
ＹおよびＹ’は、同一または異なって、〔３２〕記載の式（Ｂ－１）のＹと同じであり、
ＺおよびＺ’は、同一または異なって、前記式（Ｂ－１）のＺと同じである。〕で表される、〔３９〕のいずれかの化合物またはその塩。
〔４１〕下記式（Ｉ）：
Ａ－Ｌ－Ｂ－Ｒ （Ｉ）
〔式中、
Ａは、可溶性タンパク質に対する親和性物質であり、
Ｌは、切断性部分を含む２価の基である切断性リンカーであり、
Ｂは、（ａ）生体直交性官能基を含む２価の基、または（ｂ）生体直交性官能基を含まない２価の基であり、
Ｒは、前記可溶性タンパク質に対する反応性基である。〕で表される、可溶性タンパク質に対する親和性物質、切断性部分および反応性基を有する化合物またはその塩を含む、可溶性タンパク質の位置選択的修飾試薬。
〔４２〕下記式（Ｉ）：
Ａ－Ｌ－Ｂ－Ｒ （Ｉ）
〔式中、
Ａは、抗体に対する親和性物質であり、
Ｌは、切断性部分を含む２価の基である切断性リンカーであり、
Ｂは、（ａ）生体直交性官能基を含む２価の基、または（ｂ）生体直交性官能基を含まない２価の基であり、
Ｒは、リジン残基の側鎖に対して特異的な反応性基である。〕で表される、抗体に対する親和性物質、切断性部分および反応性基を有する化合物またはその塩。
〔４３〕下記式（Ｉ）：
下記式（Ｉ）：
Ａ－Ｌ－Ｂ－Ｒ （Ｉ）
〔式中、
Ａは、抗体に対する親和性物質であり、
Ｌは、切断性部分を含む２価の基である切断性リンカーであり、
Ｂは、（ａ）生体直交性官能基を含む２価の基、または（ｂ）生体直交性官能基を含まない２価の基であり、
Ｒは、リジン残基の側鎖に対して特異的な反応性基である。〕で表される、抗体に対する親和性物質、切断性部分および反応性基を有する化合物またはその塩を含む、抗体の位置選択的修飾試薬。 In a first embodiment, the present invention provides a soluble protein regioselective modification reagent comprising an affinity substance for a soluble protein, a compound having a cleavable moiety and a reactive group or a salt thereof, and the compound or a salt thereof. offer.
[1] Formula (I) below:
A-L-B-R (I)
[In the formula,
A is an affinity substance for soluble proteins;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B is (a) a divalent group that includes a bioorthogonal functional group or (b) a divalent group that does not include a bioorthogonal functional group;
R is a reactive group for said soluble protein. ], a compound or a salt thereof having an affinity substance for a soluble protein, a cleavable moiety and a reactive group.
[2] L is (i) a cleavable linker that is a divalent group comprising a cleavable moiety capable of generating a bioorthogonal functional group on the reactive group side by cleavage, or (ii) a bioorthogonal The compound of [1] or a salt thereof, which is a cleavable linker that is a divalent group containing a cleavable moiety that does not have the ability to generate a functional group on the reactive group side.
[3] The compound of [2] or a salt thereof, wherein L is the cleavable linker of (i).
[4] The compound or salt of [2] or [3], wherein L is the cleavable linker of (i) above and B is the divalent group of (b) above.
[5] The compound of [2] or a salt thereof, wherein L is the cleavable linker of (ii) above and B is the divalent group of (a) above.
[6] The compound or salt of any one of [1] to [5], wherein the substance with affinity for soluble proteins is a peptide.
[7] The compound of [6] or a salt thereof, wherein the peptide is a binding peptide to the Fc region of a monoclonal antibody.
[8] The compound of [7] or a salt thereof, wherein the binding peptide is a binding peptide to the Fc region of IgG.
[9] the affinity substance is an affinity substance for an antibody containing any one Fc region protein selected from the group consisting of the following (A) to (C) and having antigen-binding ability [1] to any one of [8] or a salt thereof:
(A) an Fc region protein comprising the amino acid sequence of SEQ ID NO: 1;
(B) an Fc region protein comprising an amino acid sequence in which one or several amino acid residues are inserted, added, deleted or substituted in the amino acid sequence of SEQ ID NO: 1; or (C) the amino acid sequence of SEQ ID NO: 1 and 90 An Fc region protein comprising an amino acid sequence exhibiting greater than or equal to % identity.
[10] The binding peptide has the following formula (i):
(X _1-3 )-C-(X ₂ )-H-(Xaa1)-G-(Xaa2)-LVWC-(X _1-3 ) (SEQ ID NO: 94) (i)
[In the formula,
X is the same or different and is any amino acid residue other than cysteine;
C is a cysteine residue,
H is a histidine residue,
Xaa1 is an arginine residue, a leucine residue, a lysine residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue;
G is a glycine residue,
Xaa2 is a lysine residue, a glutamine residue, a glutamic acid residue, an asparagine residue, or an aspartic acid residue;
L is a leucine residue,
V is a valine residue and W is a tryptophan residue. ]
The compound of any one of [7] to [9], which is a peptide or a salt thereof comprising an amino acid sequence consisting of 13 to 17 amino acid residues represented by and capable of binding to human IgG and/or rabbit IgG. Or its salt.
[11] the binding peptide is represented by the following formula (i-1):
(X _1-3 )-C-(X ₂ )-H-(Xaa1)-G-(Xaa2)-LVWC-(X _1-3 ) (SEQ ID NO: 95) (i-1)
[In the formula,
X is the same or different and is any amino acid residue other than cysteine;
C is a cysteine residue,
H is a histidine residue,
Xaa1 is a lysine residue, a cysteine residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue;
G is a glycine residue,
Xaa2 is a glutamic acid residue or an asparagine residue,
L is a leucine residue,
V is a valine residue and W is a tryptophan residue. ]
The compound of any one of [7] to [9], which is a peptide or a salt thereof comprising an amino acid sequence consisting of 13 to 17 amino acid residues represented by and capable of binding to human IgG and/or rabbit IgG. Or its salt.
[12] a binding peptide represented by the following formula (i-2):
(X _1-3 )-C-(X ₂ )-H-(Xaa1)-G-(Xaa2)-LVWC-(X _1-3 ) (SEQ ID NO: 96) (i-2)
[In the formula,
X is the same or different and is any amino acid residue other than cysteine;
C is a cysteine residue,
H is a histidine residue,
Xaa1 is an arginine residue or a leucine residue,
G is a glycine residue,
Xaa2 is a lysine residue, a glutamine residue, or an aspartic acid residue;
L is a leucine residue,
V is a valine residue, and
W is a tryptophan residue. ], a peptide or a salt thereof comprising an amino acid sequence consisting of 13 to 17 amino acid residues and capable of binding to human IgG and/or rabbit IgG, any of [7] to [10] compound or its salt.
[13] The binding peptide has the following formula (v):
(X _1-3 )-C-(Xaa3)-(xaa4)-H-(Xaa1)-G-(Xaa2)-LVWC-(Xaa5)-(Xaa6)-(Xaa7) (sequence number 102)
(v)
[In the formula,
X is the same or different and is any amino acid residue other than cysteine;
C is a cysteine residue,
Xaa3 is an alanine residue or a lysine residue,
Xaa4 is a tryptophan residue or a tyrosine residue,
H is a histidine residue,
Xaa1 is an arginine residue, a leucine residue, a lysine residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue;
G is a glycine residue,
Xaa2 is a lysine residue, a glutamine residue, a glutamic acid residue, an asparagine residue, or an aspartic acid residue;
L is a leucine residue,
V is a valine residue,
W is a tryptophan residue,
Xaa5 is a threonine residue or a lysine residue,
Xaa6 is a tyrosine residue, a lysine residue, or no residue, and Xaa7 is a histidine residue, a lysine residue, or no residue. Any one of [7] to [12], which is a peptide or a salt thereof, which comprises an amino acid sequence consisting of 13 to 17 amino acid residues and is capable of binding to human IgG and/or rabbit IgG, represented by compound or its salt.
[14] The binding peptide has the following formula (vi):
DC-(Xaa3)-(Xaa4)-H-(Xaa1)-G-(Xaa2)-LVWC-(Xaa5)-(Xaa6)-(Xaa7) (SEQ ID NO: 103) (vi ) [In the formula,
D is an aspartic acid residue,
C is a cysteine residue,
Xaa3 is an alanine residue or a lysine residue,
Xaa4 is a tryptophan residue or a tyrosine residue,
H is a histidine residue,
Xaa1 is an arginine residue, a leucine residue, a lysine residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue;
G is a glycine residue,
Xaa2 is a lysine residue, a glutamine residue, a glutamic acid residue, an asparagine residue, or an aspartic acid residue;
L is a leucine residue,
V is a valine residue,
W is a tryptophan residue,
Xaa5 is a threonine residue or a lysine residue,
Xaa6 is a tyrosine residue, a lysine residue, or no residue;
Xaa7 is a histidine residue, a lysine residue, or none. ], and is capable of binding to human IgG and/or rabbit IgG, or a salt thereof, [7] to [13 ] or a salt thereof.
[15] The binding peptide is represented by the following formula (vii):
DC-(Xaa3)-(Xaa4)-H-(Xaa1)-G-(Xaa2)-LVWCT (SEQ ID NO: 104) (vii)
[In the formula,
D is an aspartic acid residue,
C is a cysteine residue,
Xaa3 is an alanine residue or a lysine residue,
Xaa4 is a tryptophan residue or a tyrosine residue,
H is a histidine residue,
Xaa1 is an arginine residue, a leucine residue, a lysine residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue;
G is a glycine residue,
Xaa2 is a lysine residue, a glutamine residue, a glutamic acid residue, an asparagine residue, or an aspartic acid residue;
L is a leucine residue,
V is a valine residue,
W is a tryptophan residue, and T is a threonine residue], and is capable of binding to human IgG and/or rabbit IgG. The compound or salt thereof according to any one of [7] to [14], which is a characterized peptide or a salt thereof.
[16] the binding peptide is represented by the following formula (viii):
RGNC-(Xaa3)-(Xaa4)-H-(Xaa1)-G-(Xaa2)-LVWC-(Xaa5)-(Xaa6)-(Xaa7) (SEQ ID NO: 105) (viii)
[In the formula,
R is an arginine residue,
G is a glycine residue,
N is an asparagine residue,
C is a cysteine residue,
Xaa3 is an alanine residue or a lysine residue,
Xaa4 is a tryptophan residue or a tyrosine residue,
H is a histidine residue,
Xaa1 is an arginine residue, a leucine residue, a lysine residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue;
G is a glycine residue,
Xaa2 is a lysine residue, a glutamine residue, a glutamic acid residue, an asparagine residue, or an aspartic acid residue;
L is a leucine residue,
V is a valine residue,
W is a tryptophan residue,
Xaa5 is a threonine residue or a lysine residue,
Xaa6 is a tyrosine residue, a lysine residue, or no residue, and Xaa7 is a histidine residue, a lysine residue, or no residue. ], and is capable of binding to human IgG and/or rabbit IgG, or a salt thereof, [7] to [13 ] or a salt thereof.
[17] The compound or salt of any one of [7] to [16], wherein the binding peptide is capable of binding to human IgG.
[18] any amino acid residue in the amino acid sequence of (a) FNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 92) in which the binding peptide is a lysine residue, an aspartic acid residue, a glutamic acid residue, or a 2-aminosuberic acid residue; , and a diaminopropionic acid residue, and (b) having 90% or more identity to said amino acid sequence of SEQ ID NO: 92. , an affinity peptide or a salt thereof, the compound of any one of [7] to [9] or a salt thereof.
[19] The cleavable moiety is (a) treated with one or more substances selected from the group consisting of acidic substances, basic substances, reducing agents, oxidizing agents, and enzymes; The moiety of any one of [1] to [18], which is cleavable either by treatment with a chemical stimulus or (c) by leaving in the case of using a cleavable linker containing a self-cleavable cleavable moiety. compound or its salt.
[20] the cleavable moiety is a disulfide residue, an acetal residue, a ketal residue, an ester residue, a carbamoyl residue, an alkoxyalkyl residue, an imine residue, a tertiary alkyloxycarbamate residue, a silane residue; hydrazone-containing residues, phosphoramidate residues, aconityl residues, trityl residues, azo residues, vicinal diol residues, selenium residues, aromatic ring-containing residues with electron-withdrawing groups, coumarin-containing residues , a sulfone-containing residue, an unsaturated bond-containing chain residue, and a glycosyl residue, the compound or a salt thereof according to any one of [1] to [19].
[21] The cleavable moiety of (i) is selected from the group consisting of disulfide residues, ester residues, acetal residues, ketal residues, imine residues, and vicinal diol residues, [2] to [ 20] or a salt thereof.
[22] The cleavable moiety of (ii) is an ester residue, a carbamoyl residue, an alkoxyalkyl residue, an imine residue, a tertiary alkyloxycarbamate residue, a silane residue, a hydrazone-containing residue, a phosphorami date residue, aconityl residue, trityl residue, azo residue, vicinal diol residue, selenium residue, aromatic ring-containing residue with electron-withdrawing group, coumarin-containing residue, sulfone-containing residue, unsaturated The compound or salt thereof according to any one of [2] to [20], which is selected from the group consisting of bond-containing chain residues and glycosyl residues.
[23] the cleavable moiety comprises:

[Where the wavy line perpendicular to the bond indicates the cleavage site,
multiple R _2a , multiple R _2b , and multiple R _2c are the same or different,
(i) a hydrogen atom, or a halogen atom;
(ii) a monovalent hydrocarbon group;
(iii) aralkyl;
(iv) a monovalent heterocyclic group;
(v) R _c -O-, R _c -C(=O)-, R _c -O-C(=O)-, or R _c -C(=O)-O- (R _c is a hydrogen atom , or a monovalent hydrocarbon group);
(vi) NR _d R _e -, NR _d R _e -C(=O)-, NR _d R _e -C(=O)-O-, or R _d -C(=O)-NR _e -(R _d and R _e are the same or different and represent a hydrogen atom or a monovalent hydrocarbon group); or (vii) from the group consisting of a nitro group, a sulfate group, a sulfonate group, a cyano group, or a carboxyl group chosen,
J is —CH ₂ —, —O—, or —S—;
r is any integer from 1 to 4,
O (white circle) indicates binding to A, and ● (filled circle) indicates binding to B.
If the chemical structure is asymmetric about the cleavage site, the circle may indicate the bond to A and the circle may indicate the bond to B. ] corresponding to any one chemical structure selected from the group consisting of [1] to [20] or a salt thereof.
[24] The cleavable moiety of (i) above is:

[Where the wavy line perpendicular to the bond indicates the cleavage site,
R _2a is the same as [23],
O (white circle) indicates binding to A, and ● (filled circle) indicates binding to B.
If the chemical structure is asymmetric about the cleavage site, the circle may indicate the bond to A and the circle may indicate the bond to B. ] corresponding to any one chemical structure selected from the group consisting of [2] to [19], [21], [23] or a salt thereof.
[25] The cleavable moiety of (ii) above is:

[Where the wavy line perpendicular to the bond indicates the cleavage site,
R _2b , R _2c , J and r are the same as in [23],
O (white circle) indicates binding to A, and ● (filled circle) indicates binding to B.
If the chemical structure is asymmetric about the cleavage site, the circle may indicate the bond to A and the circle may indicate the bond to B. ] corresponding to any one chemical structure selected from the group consisting of [2] to [19], [22], [23] or a salt thereof.
[26] L is represented by the following formulas (L1) to (L3):
La-C-Lb (L1)
La-C (L2)
C-Lb (L3)
[In the formula,
La and Lb are each a divalent group,
C is a cleavable moiety. ], the compound or a salt thereof according to any one of [1] to [25].
[27] La and Lb are respectively the following (La') and (Lb'):

[In the formula,
p and p' are the same or different and any integer from 0 to 10;
q and q' are the same or different and any integer from 0 to 10;
X and X' are the same or different and are a carbon atom, a nitrogen atom, or a single bond (wherein when X is a nitrogen atom, R _1b is absent, and when X' is a nitrogen atom, R _{1b '} is absent.If X is a single bond, R _1a and R _1b are absent, and if X' is a single bond, R _1a' and R _1b' are absent);
R _1a , R _1b , R _1a′ and R _1b′ are the same or different and are atoms or groups selected from the group consisting of (i) to (vii). ], the compound of [26] or a salt thereof.
[28] the bivalent group containing a bioorthogonal functional group is an azide residue, an aldehyde residue, a thiol residue, an alkyne residue, an alkene residue, a tetrazine residue, a nitrone residue, a hydroxylamine residue, a nitrile residue, hydrazine residue, ketone residue, boronic acid residue, cyanobenzothiazole residue, allyl residue, phosphine residue, maleimide residue, disulfide residue, thioester group, α-halocarbonyl residue, isonitrile residue The compound or a salt thereof according to any one of [1] to [27], which is a divalent group containing a bioorthogonal functional group selected from the group consisting of groups, sydnon residues and selenium residues in its main chain.
[29] the bivalent group containing a bioorthogonal functional group is an azide residue, an aldehyde residue, a thiol residue, an alkyne residue, an alkene residue, a halogen residue, a tetrazine residue, a nitrone residue, hydroxylamine residue, nitrile residue, hydrazine residue, ketone residue, boronic acid residue, cyanobenzothiazole residue, allyl residue, phosphine residue, maleimide residue, disulfide residue, α-halocarbonyl residue, isonitrile The compound or a salt thereof according to any one of [1] to [27], which is a divalent group containing a bioorthogonal functional group selected from the group consisting of a residue, a sydnon residue and a selenium residue in its side chain.
[30] The bioorthogonal functional group is:

[In the formula,
R _1f , single or multiple R _1g and single or multiple R _1h are the same or different and are atoms or groups selected from the group consisting of (i) to (vii), or electron-withdrawing groups;
・ is a bond. ), the compound or a salt thereof according to any one of [1] to [29].
[31] The divalent group (b) is optionally substituted alkylene, optionally substituted cycloalkylene, optionally substituted aryl, or optionally substituted divalent heterocyclic ring group, -NR _a - (R _a represents a hydrogen atom or a substituent), -O-, or a compound of any one of [1] to [30] selected from the group consisting of a combination of two or more thereof Or its salt.
[32] B is the following formula (B-1):

[In the formula,
Y is -NH-, -O-, -CH ₂ -, or the following formula (B-2):

(In the formula,
V and V′ are the same or different and are —NH—, —O—, —CH ₂ —, or a single bond;
V is a divalent group containing a bioorthogonal functional group;
s is any integer from 0 to 10,
○ and ● in formula (B-2) have the same orientations as ○ and ● in formula (B-1), respectively. ) and
Z is an oxygen atom, a sulfur atom, or a hydrogen atom (when Z is a hydrogen atom, -C(=Z)- represents -CH ₂ -);
○ (white circle) in formula (B-1) indicates a bond to the L-side portion, and ● (black circle) indicates a bond to the R-side portion. ], the compound or a salt thereof according to any one of [1] to [31].
[33] any one of [1] to [32], wherein the reactive group is a reactive group specific to the side chain of any one of a lysine residue, a tyrosine residue, or a tryptophan residue; compound or its salt.
[34] the compound of [33] or a salt thereof, wherein the reactive group is a reactive group specific to the side chain of a lysine residue;
[35] The reactive group is the following:

〔here,
R _5a and R _5c are atoms or groups selected from the group consisting of (i) to (vii);
R _5b is an electron withdrawing group,
j is any integer from 1 to 5;
k is any integer from 1 to 4. ] corresponding to any one chemical structure selected from the group consisting of [1] to [34] or a salt thereof.
[36] The compound or salt of any one of [1] to [35], wherein the main chain connecting A and R has 4 to 20 atoms.
[37] The compound or a salt thereof according to any one of [1] to [36], wherein the main chain connecting A and R does not contain a ring structure.
[38] The compound or a salt thereof according to any one of [1] to [37], wherein the partial structure represented by LB does not contain a peptide moiety.
[39] The compound represented by the formula (I) is the following (I'):
AB2-L'-B1-R (I')
[In the formula,
A and R are the same as in formula (I) above L′ is a cleavable linker that is a divalent group containing a cleavable moiety;
B1 and B2 are the same or different and are (a) a divalent group containing a bioorthogonal functional group or (b) a divalent group without a bioorthogonal functional group;
B1 and B2 may have a symmetrical structure about L'. ], the compound or a salt thereof according to any one of [1] to [38].
[40] The compound represented by the formula (I′) is the following (I″):

[In the formula,
A and R are the same as those of formula (I) described in [1],
C is a cleavable moiety;
p, p', q, q', X, X', R _1a , R _1a' , R _1b , and R _1b' are the same as those of formulas (La') and (Lb') described in [27] and
Y and Y' are the same or different and are the same as Y in formula (B-1) described in [32],
Z and Z' are the same or different and are the same as Z in formula (B-1) above. ], any one of the compounds of [39] or a salt thereof.
[41] the following formula (I):
A-L-B-R (I)
[In the formula,
A is an affinity substance for soluble proteins;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B is (a) a divalent group that includes a bioorthogonal functional group or (b) a divalent group that does not include a bioorthogonal functional group;
R is a reactive group for said soluble protein. ], a regioselective modification reagent for a soluble protein comprising a compound having an affinity for a soluble protein, a cleavable moiety and a reactive group or a salt thereof.
[42] the following formula (I):
A-L-B-R (I)
[In the formula,
A is an affinity substance for the antibody;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B is (a) a divalent group that includes a bioorthogonal functional group or (b) a divalent group that does not include a bioorthogonal functional group;
R is a reactive group specific for the side chains of lysine residues. ], a compound or a salt thereof having an affinity substance for an antibody, a cleavable moiety and a reactive group.
[43] the following formula (I):
Formula (I) below:
A-L-B-R (I)
[In the formula,
A is an affinity substance for the antibody;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B is (a) a divalent group that includes a bioorthogonal functional group or (b) a divalent group that does not include a bioorthogonal functional group;
R is a reactive group specific for the side chains of lysine residues. ], an antibody regioselective modification reagent comprising an antibody-affinity substance, a cleavable moiety and a compound having a reactive group or a salt thereof.

〔ここで、●（黒丸）は、Ｔ側の部分に対する結合を示し、○（白丸）は、Ｂ側の部分に対する結合を示す。結合に直交する直線は、反応により生成する結合を示す。〕からなる群より選ばれるいずれか一つの化学構造に対応する、〔４４〕～〔６１〕のいずれかの可溶性タンパク質またはその塩。
〔６３〕ＡおよびＲ’を連結する主鎖の原子数が４～２０個である、〔４４〕～〔６２〕のいずれかの可溶性タンパク質またはその塩。
〔６４〕ＡおよびＲを連結する主鎖が環構造を含まない、〔４４〕～〔５３〕のいずれかの可溶性タンパク質またはその塩。
〔６５〕Ｌ－Ｂで表される部分構造がペプチド部分を含まない、〔４４〕～〔６４〕のいずれかの可溶性タンパク質またはその塩。
〔６６〕前記式（ＩＩ）で表される化合物が、下記（ＩＩ’）：
Ａ－Ｂ２－Ｌ’－Ｂ１－Ｒ’－Ｔ （ＩＩ’）
〔式中、
Ａ、Ｒ’およびＴは、前記式（ＩＩ）のものと同じであり
Ｌ’は、切断性部分を含む２価の基である切断性リンカーであり、
Ｂ１およびＢ２は、同一または異なって、（ａ）生体直交性官能基を含む２価の基、または（ｂ）生体直交性官能基を含まない２価の基であり、
Ｂ１およびＢ２は、Ｌ’を中心にした対称構造を有していてもよい。〕で表される化合物である、〔４４〕～〔６５〕のいずれかの可溶性タンパク質またはその塩。
〔６７〕前記式（ＩＩ’）で表される化合物が、下記（ＩＩ’’）：

〔式中、
Ａ、Ｒ’およびＴは、〔４４〕記載の式（ＩＩ）のものと同じであり、
Ｃは、切断性部分であり、
ｐおよびｐ’は、同一または異なって、０～１０の任意の整数であり、
ｑおよびｑ’は、同一または異なって、０～１０の任意の整数であり、
ＸおよびＸ’は、同一または異なって、炭素原子、窒素原子、または単結合（ここで、Ｘが窒素原子である場合、Ｒ_１ｂは存在せず、Ｘ’が窒素原子である場合、Ｒ_１ｂ’は存在しない。Ｘが単結合である場合、Ｒ_１ａおよびＲ_１ｂは存在せず、Ｘ’が単結合である場合、Ｒ_１ａ’およびＲ_１ｂ’は存在しない）であり、
Ｒ_１ａ、Ｒ_１ｂ、Ｒ_１ａ’およびＲ_１ｂ’は、同一または異なって、
（ｉ）水素原子、またはハロゲン原子；
（ｉｉ）１価の炭化水素基；
（ｉｉｉ）アラルキル；
（ｉｖ）１価の複素環基；
（ｖ）Ｒ_ｃ－Ｏ－、Ｒ_ｃ－Ｃ（＝Ｏ）－、Ｒ_ｃ－Ｏ－Ｃ（＝Ｏ）－、もしくはＲ_ｃ－Ｃ（＝Ｏ）－Ｏ－（Ｒ_ｃは、水素原子、もしくは一価の炭化水素基を示す。）；
（ｖｉ）ＮＲ_ｄＲ_ｅ－、ＮＲ_ｄＲ_ｅ－Ｃ（＝Ｏ）－、ＮＲ_ｄＲ_ｅ－Ｃ（＝Ｏ）－Ｏ－、もしくはＲ_ｄ－Ｃ（＝Ｏ）－ＮＲ_ｅ－（Ｒ_ｄおよびＲ_ｅは、同一もしくは異なって、水素原子、もしくは一価の炭化水素基を示す。）；または
（ｖｉｉ）ニトロ基、硫酸基、スルホン酸基、シアノ基、もしくはカルボキシル基
からなる群より選ばれ、
ＹおよびＹ’は、同一または異なって、〔３２〕記載の式（Ｂ－１）のＹと同じであり、
ＺおよびＺ’は、同一または異なって、前記式（Ｂ－１）のＺと同じである。〕で表さ
れる、〔６６〕の可溶性タンパク質またはその塩。
〔６８〕下記式（ＩＩ）：
Ａ－Ｌ－Ｂ－Ｒ’－Ｔ （ＩＩ）
〔式中、
Ａは、抗体に対する親和性物質であり、
Ｌは、切断性部分を含む２価の基である切断性リンカーであり、
Ｂは、（ａ）生体直交性官能基を含む２価の基、または（ｂ）生体直交性官能基を含まない２価の基であり、
Ｒ’は、抗体と、リジン残基の側鎖に対して特異的な反応性基との間の反応により生成する部分であり、
Ｔは、抗体である。〕表される、抗体に対する親和性物質、および切断性部分を有する抗体またはその塩。 Second, the present invention provides affinity substances for soluble proteins and soluble proteins or salts thereof having cleavable moieties, and methods for their production.
(Affinity Substances for Soluble Proteins and Soluble Proteins with Cleavable Moieties or Salts thereof)
[44] Formula (II) below:
A-LB-R'-T (II)
[In the formula,
A is an affinity substance for soluble proteins;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B is (a) a divalent group that includes a bioorthogonal functional group or (b) a divalent group that does not include a bioorthogonal functional group;
R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein. ] and a soluble protein having a cleavable moiety or a salt thereof.
[45] The soluble protein of [44] or a salt thereof, wherein the soluble protein is a monoclonal antibody.
[46] The soluble protein of [44] or [45], wherein the soluble protein is an IgG antibody, or a salt thereof.
[47] The soluble protein or salt thereof of any one of [44] to [46], wherein the soluble protein is human-derived.
[48] any one of [44] to [47], wherein the soluble protein comprises any one Fc region protein selected from the group consisting of the following (A) to (C) and is an antibody having antigen-binding ability: Soluble protein or salt thereof:
(A) an Fc region protein comprising the amino acid sequence of SEQ ID NO: 1;
(B) an Fc region protein comprising an amino acid sequence in which one or several amino acid residues are inserted, added, deleted or substituted in the amino acid sequence of SEQ ID NO: 1; or (C) the amino acid sequence of SEQ ID NO: 1 and 90 An Fc region protein comprising an amino acid sequence exhibiting greater than or equal to % identity.
[49] the soluble protein contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and the specific amino acid in a non-target region other than the target region containing 5 or more residues,
The structural unit represented by ALBR' binds to one or more specific amino acid residues contained in the target region with a regioselectivity of 30% or more [44 ] to [48] any soluble protein or a salt thereof.
[50] The soluble protein or salt thereof of [49], wherein the target region is a region consisting of 1 to 10 consecutive amino acid residues.
[51] The soluble protein of [50] or a salt thereof, wherein the target region is a region consisting of 1 to 3 consecutive amino acid residues.
[52] The target region is (a) a region consisting of amino acid residues at positions 246-248 in the human IgG Fc region, (b) a region consisting of amino acid residues at positions 288-290 in the human IgG Fc region, or ( c) the soluble protein of [51], which is a region consisting of amino acid residues at position 317 in the human IgG Fc region, or a salt thereof.
[53] The soluble protein or salt thereof according to any one of [49] to [52], wherein the regioselectivity is 50% or more.
[54] The soluble protein of [53] or a salt thereof, wherein the regioselectivity is 70% or more.
[55] The soluble protein of [54] or a salt thereof, wherein the regioselectivity is 90% or more.
[56] the specific amino acid residue is a (where a is any integer from 1 to 10 ) does not contain amino acid residues of the same kind as the specific amino acid residue other than the specific amino acid residue present at the specific position in the region up to the remote position of the amino acid residue of [49] to [ 55] any soluble protein or a salt thereof.
[57] the soluble protein is a multimeric protein comprising a plurality of monomeric proteins;
T has a structural unit represented by ALBR' in a plurality of corresponding target regions in a plurality of monomeric proteins, so that the multimeric protein is ALB- The soluble protein or salt thereof according to any one of [44] to [56], which has a plurality of structural units represented by R'.
[58] the soluble protein is an antibody comprising a plurality of heavy chains;
T has a structural unit represented by ALBR' in a plurality of corresponding target regions in a plurality of heavy chains, such that the antibody is represented by ALBR'. The soluble protein or salt thereof according to any one of [44] to [57], which has a plurality of structural units of
[59] The soluble protein of [58] or a salt thereof, wherein the number of heavy chains is two.
[60] the portion generated by the reaction between the soluble protein and the reactive group is any one of a lysine residue, a tyrosine residue, or a tryptophan residue for a lysine residue, a tyrosine residue, or a tryptophan residue; The soluble protein or salt thereof according to any one of [44] to [59], which is a moiety produced by reaction of a reactive group specific to one side chain.
[61] the moiety generated by the reaction between the soluble protein and the reactive group is generated by the reaction between a lysine residue and a reactive group specific for the side chain of the lysine residue; A soluble protein or a salt thereof according to any one of [44] to [60].
[62] The moiety produced by the reaction is:

[Here, ● (black circle) indicates a bond to the portion on the T side, and O (white circle) indicates a bond to the portion on the B side. A straight line perpendicular to the bond indicates the bond produced by the reaction. ] the soluble protein or a salt thereof according to any one of [44] to [61], which corresponds to any one chemical structure selected from the group consisting of:
[63] The soluble protein or salt thereof of any one of [44] to [62], wherein the main chain connecting A and R' has 4 to 20 atoms.
[64] The soluble protein or salt thereof of any one of [44] to [53], wherein the main chain connecting A and R does not contain a ring structure.
[65] The soluble protein or salt thereof of any one of [44] to [64], wherein the partial structure represented by LB does not contain a peptide moiety.
[66] The compound represented by the formula (II) is the following (II'):
AB2-L'-B1-R'-T (II')
[In the formula,
A, R′ and T are the same as in formula (II) above L′ is a cleavable linker that is a divalent group containing a cleavable moiety;
B1 and B2 are the same or different and are (a) a divalent group containing a bioorthogonal functional group or (b) a divalent group containing no bioorthogonal functional group;
B1 and B2 may have a symmetrical structure about L'. ], a soluble protein or a salt thereof according to any one of [44] to [65].
[67] The compound represented by the formula (II') is the following (II''):

[In the formula,
A, R' and T are the same as those of formula (II) described in [44],
C is a cleavable moiety;
p and p' are the same or different and any integer from 0 to 10;
q and q' are the same or different and any integer from 0 to 10;
X and X' are the same or different and are a carbon atom, a nitrogen atom, or a single bond (wherein when X is a nitrogen atom, R _1b is absent, and when X' is a nitrogen atom, R _{1b '} is absent.If X is a single bond, R _1a and R _1b are absent, and if X' is a single bond, R _1a' and R _1b' are absent);
R _1a , R _1b , R _1a′ and R _1b′ are the same or different,
(i) a hydrogen atom, or a halogen atom;
(ii) a monovalent hydrocarbon group;
(iii) aralkyl;
(iv) a monovalent heterocyclic group;
(v) R _c -O-, R _c -C(=O)-, R _c -O-C(=O)-, or R _c -C(=O)-O- (R _c is a hydrogen atom , or a monovalent hydrocarbon group);
(vi) NR _d R _e -, NR _d R _e -C(=O)-, NR _d R _e -C(=O)-O-, or R _d -C(=O)-NR _e -(R _d and R _e are the same or different and represent a hydrogen atom or a monovalent hydrocarbon group); or (vii) from the group consisting of a nitro group, a sulfate group, a sulfonate group, a cyano group, or a carboxyl group chosen,
Y and Y' are the same or different and are the same as Y in formula (B-1) described in [32],
Z and Z' are the same or different and are the same as Z in formula (B-1) above. ], the soluble protein of [66] or a salt thereof.
[68] the following formula (II):
A-LB-R'-T (II)
[In the formula,
A is an affinity substance for the antibody;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B is (a) a divalent group that includes a bioorthogonal functional group or (b) a divalent group that does not include a bioorthogonal functional group;
R' is a moiety generated by reaction between an antibody and a reactive group specific for the side chain of a lysine residue;
T is an antibody. ] and an antibody or salt thereof having a cleavable moiety, represented by an affinity substance for an antibody.

(Affinity Substance for Soluble Protein and Method for Producing Soluble Protein Having Cleavable Portion or Salt thereof)
[69] A method for producing a soluble protein having an affinity for a soluble protein and a soluble protein having a cleavable moiety or a salt thereof,
Formula (I) below:
A-L-B-R (I)
[In the formula,
A is an affinity substance for soluble proteins;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B is (a) a divalent group that includes a bioorthogonal functional group or (b) a divalent group that does not include a bioorthogonal functional group;
R is a reactive group for said soluble protein. ], a compound having an affinity for soluble proteins, a cleavable moiety and a reactive group or a salt thereof, is reacted with a soluble protein,
Formula (II) below:
A-LB-R'-T (II)
[In the formula,
A, L, and B are the same as in formula (I) above;
R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein. and a soluble protein or salt thereof having a cleavable moiety.
[70] The method of [69], wherein the soluble protein is an antibody and the reactive group is a reactive group specific to the side chain of a lysine residue.

〔ここで、結合に直交する波線は、反応により生成する結合を示し、
複数のＲ_２ａ、複数のＲ_２ｂ、および複数のＲ_２ｃは、同一または異なって、
（ｉ）水素原子、またはハロゲン原子；
（ｉｉ）１価の炭化水素基；
（ｉｉｉ）アラルキル；
（ｉｖ）１価の複素環基；
（ｖ）Ｒ_ｃ－Ｏ－、Ｒ_ｃ－Ｃ（＝Ｏ）－、Ｒ_ｃ－Ｏ－Ｃ（＝Ｏ）－、もしくはＲ_ｃ－Ｃ（＝Ｏ）－Ｏ－（Ｒ_ｃは、水素原子、もしくは一価の炭化水素基を示す。）；
（ｖｉ）ＮＲ_ｄＲ_ｅ－、ＮＲ_ｄＲ_ｅ－Ｃ（＝Ｏ）－、ＮＲ_ｄＲ_ｅ－Ｃ（＝Ｏ）－Ｏ－、もしくはＲ_ｄ－Ｃ（＝Ｏ）－ＮＲ_ｅ－（Ｒ_ｄおよびＲ_ｅは、同一もしくは異なって、水素原子、もしくは一価の炭化水素基を示す。）；または
（ｖｉｉ）ニトロ基、硫酸基、スルホン酸基、シアノ基、もしくはカルボキシル基
からなる群より選ばれ、
Ｊは、－ＣＨ_２－、－Ｏ－、または－Ｓ－であり、
ｒは、１～４の任意の整数であり、
○（白丸）はＡに対する結合を示し、●（黒丸）はＢに対する結合を示す。
化学構造が、切断部位を中心にして非対称である場合、●がＡに対する結合を示し、○がＢに対する結合を示していてもよい。〕からなる群より選ばれるいずれか一つの化学構造に対応する、〔７１〕～〔８０〕のいずれかの複合体またはその塩。
〔８２〕可溶性タンパク質と反応性基との間の反応により生成する部分が、リジン残基、チロシン残基、またはトリプトファン残基と、リジン残基、チロシン残基、またはトリプトファン残基のいずれか一つの側鎖に対して特異的な反応性基との間の反応により生成する部分である、〔７１〕～〔８１〕のいずれかの複合体またはその塩。
〔８３〕可溶性タンパク質と反応性基との間の反応により生成する部分が、リジン残基と
、リジン残基の側鎖に対して特異的な反応性基との間の反応により生成する部分である、〔７１〕～〔８２〕のいずれかの複合体またはその塩。
〔８４〕前記反応により生成する部分が、下記：

〔ここで、●（黒丸）は、Ｔ側の部分に対する結合を示し、○（白丸）は、Ｂ側の部分に対する結合を示す。〕からなる群より選ばれるいずれか一つの化学構造に対応する、〔７１〕～〔８３〕のいずれかの複合体またはその塩。
〔８５〕機能性物質が薬物または標識物質である、〔７１〕～〔８４〕のいずれかの複合体またはその塩。
〔８６〕機能性物質が低分子化合物である、〔７１〕～〔８５〕のいずれかの複合体またはその塩。
〔８７〕薬物が抗癌剤である、〔８５〕または〔８６〕の複合体またはその塩。
〔８８〕ＡおよびＲ’を連結する主鎖の原子数が４～２０個である、〔７１〕～〔８７〕のいずれかの複合体またはその塩。
〔８９〕ＡおよびＲを連結する主鎖が環構造を含まない、〔７１〕～〔８８〕のいずれかの複合体またはその塩。
〔９０〕Ｌ－Ｂで表される部分構造がペプチド部分を含まない、〔７１〕～〔８９〕のいずれかの複合体またはその塩。
〔９１〕前記式（ＩＩＩ）で表される化合物が、下記式（ＩＩＩ’）：
Ａ－Ｂ２’（－Ｆ２）－Ｌ’－Ｂ１’（－Ｆ１）－Ｒ’－Ｔ （ＩＩＩ’）
〔式中、
Ａ、Ｒ’およびＴは、前記式（ＩＩ）のものと同じであり、
Ｌ’は、切断性部分を含む２価の基である切断性リンカーであり、
Ｂ１’およびＢ２’は、同一または異なって、機能性物質と生体直交性官能基との間の反応により生成する部分を含む２価の基であり、
Ｆ１およびＦ２は、同一または異なって、機能性物質であり、
Ｂ１’（－Ｆ１）およびＢ２’（－Ｆ２）は、Ｌ’を中心にした対称構造を有していてもよい。〕で表される、〔７１〕～〔９０〕のいずれかの複合体またはその塩。
〔９２〕前記式（ＩＩＩ’）で表される化合物が、下記（ＩＩＩ’’）：

〔式中、
Ａ、Ｒ’およびＴは、〔７１〕記載の式（ＩＩＩ）のものと同じであり、
Ｃは、切断性部分であり、
ｐおよびｐ’は、同一または異なって、０～１０の任意の整数であり、
ｑおよびｑ’は、同一または異なって、０～１０の任意の整数であり、
ＸおよびＸ’は、同一または異なって、炭素原子、窒素原子、または単結合（ここで、Ｘが窒素原子である場合、Ｒ_１ｂは存在せず、Ｘ’が窒素原子である場合、Ｒ_１ｂ’は存在しない。Ｘが単結合である場合、Ｒ_１ａおよびＲ_１ｂは存在せず、Ｘ’が単結合である場合、Ｒ_１ａ’およびＲ_１ｂ’は存在しない）であり、
Ｒ_１ａ、Ｒ_１ｂ、Ｒ_１ａ’およびＲ_１ｂ’は、同一または異なって、前記（ｉ）～（ｖｉｉ）からなる群より選ばれ、
ＹおよびＹ’は、同一または異なって、〔３２〕記載の式（Ｂ－１）のＹから水素原子を一つ除いた残基であり、
ＺおよびＺ’は、同一または異なって、前記式（Ｂ－１）のＺと同じであり、
ＦおよびＦ’は、同一または異なって、機能性物質である。〕で表される、〔９１〕の複合体またはその塩。
〔９３〕下記式（ＩＩＩ）：
Ａ－Ｌ－Ｂ’（－Ｆ）－Ｒ’－Ｔ （ＩＩＩ）
〔式中、
Ａは、抗体に対する親和性物質であり、
Ｌは、切断性部分を含む２価の基である切断性リンカーであり、
Ｂ’は、機能性物質と生体直交性官能基との間の反応により生成する部分を含む２価の基であり、
Ｆは、機能性物質であり、
Ｒ’は、抗体と、リジン残基の側鎖に対して特異的な反応性基との間の反応により生成する部分であり、
Ｔは、抗体である。〕で表される、親和性物質、機能性物質および抗体を有する複合体またはその塩。 Thirdly, the present invention provides a conjugate or a salt thereof having an affinity substance for soluble protein, a cleavable moiety, a functional substance and a soluble protein, and a method for producing the same.
(Complex or salt thereof having affinity substance for soluble protein, cleavable moiety, functional substance and soluble protein)
[71] the following formula (III):
ALB'(-F)-R'-T (III)
[In the formula,
A is an affinity substance for soluble proteins;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B' is a divalent group that includes a moiety generated by a reaction between a functional agent and a bioorthogonal functional group;
F is a functional substance,
R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein. ], a complex having an affinity substance for a soluble protein, a cleavable moiety, a functional substance and a soluble protein, or a salt thereof.
[72] the conjugate or salt thereof of [71], wherein the soluble protein is a monoclonal antibody;
[73] the conjugate or salt thereof of [71] or [72], wherein the soluble protein is an IgG antibody;
[74] The complex or salt thereof according to any one of [71] to [73], wherein the soluble protein is derived from humans.
[75] any one of [71] to [74], wherein the soluble protein comprises any one Fc region protein selected from the group consisting of the following (A) to (C) and is an antibody having antigen-binding ability: A complex of or salts thereof:
(A) an Fc region protein comprising the amino acid sequence of SEQ ID NO: 1;
(B) an Fc region protein comprising an amino acid sequence in which one or several amino acid residues are inserted, added, deleted or substituted in the amino acid sequence of SEQ ID NO: 1; or (C) the amino acid sequence of SEQ ID NO: 1 and 90 An Fc region protein comprising an amino acid sequence exhibiting greater than or equal to % identity.
[76] the soluble protein contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and the specific amino acid in a non-target region other than the target region; containing 5 or more residues,
The structural unit represented by ALB'(-F)-R' binds to one or more specific amino acid residues contained in the target region with a position selectivity of 30% or more. The complex or salt thereof according to any one of [71] to [75].
[77] the specific amino acid residue is a (where a is an arbitrary integer of 1 to 10 ) does not contain amino acid residues of the same kind as the specific amino acid residue other than the specific amino acid residue present at the specific position in the region to the remote position of the amino acid residue of [76] body or its salt.
[78] the soluble protein is a multimeric protein comprising a plurality of monomeric proteins;
T has a structural unit represented by ALB'(-F)-R' in a plurality of corresponding target regions in a plurality of monomeric proteins. As a result, the multimeric protein is A The complex or salt thereof according to any one of [71] to [77], which has a plurality of structural units represented by -LB'(-F)-R'.
[79] the soluble protein is an antibody comprising a plurality of heavy chains;
T has a structural unit represented by ALB'(-F)-R' in a plurality of corresponding target regions in a plurality of heavy chains, such that the antibody has ALB The complex or salt thereof according to any one of [71] to [78], having a plurality of structural units represented by '(-F)-R'.
[80] the divalent group containing the portion generated by the reaction between the functional substance and the bioorthogonal functional group is disulfide residue, acetal residue, ketal residue, ester residue, carbamoyl residue, alkoxy alkyl residues, imine residues, tertiary alkyloxycarbamate residues, silane residues, hydrazone-containing residues, phosphoramidate residues, aconityl residues, trityl residues, azo residues, vicinal diol residues, Bivalent containing a reactive moiety selected from the group consisting of selenium residues, aromatic ring-containing residues having electron withdrawing groups, coumarin-containing residues, sulfone-containing residues, unsaturated bond-containing chain residues, and glycosyl residues A complex or a salt thereof according to any one of [71] to [79], which is a group.
[81] The moiety produced by the reaction is:

[Here, the wavy line orthogonal to the bond indicates the bond generated by the reaction,
multiple R _2a , multiple R _2b , and multiple R _2c are the same or different,
(i) a hydrogen atom, or a halogen atom;
(ii) a monovalent hydrocarbon group;
(iii) aralkyl;
(iv) a monovalent heterocyclic group;
(v) R _c -O-, R _c -C(=O)-, R _c -O-C(=O)-, or R _c -C(=O)-O- (R _c is a hydrogen atom , or a monovalent hydrocarbon group);
(vi) NR _d R _e -, NR _d R _e -C(=O)-, NR _d R _e -C(=O)-O-, or R _d -C(=O)-NR _e -(R _d and R _e are the same or different and represent a hydrogen atom or a monovalent hydrocarbon group); or (vii) from the group consisting of a nitro group, a sulfate group, a sulfonate group, a cyano group, or a carboxyl group chosen,
J is —CH ₂ —, —O—, or —S—;
r is any integer from 1 to 4,
O (white circle) indicates binding to A, and ● (filled circle) indicates binding to B.
If the chemical structure is asymmetric about the cleavage site, the circle may indicate the bond to A and the circle may indicate the bond to B. ] corresponding to any one chemical structure selected from the group consisting of any one of [71] to [80] or a salt thereof.
[82] the moiety generated by the reaction between the soluble protein and the reactive group is any one of a lysine residue, a tyrosine residue, or a tryptophan residue and a lysine residue, a tyrosine residue, or a tryptophan residue; The conjugate of any one of [71] to [81] or a salt thereof, which is a moiety produced by reaction with a reactive group specific for one side chain.
[83] the moiety generated by the reaction between the soluble protein and the reactive group is generated by the reaction between a lysine residue and a reactive group specific for the side chain of the lysine residue; A complex or a salt thereof according to any one of [71] to [82].
[84] The moiety produced by the reaction is:

[Here, ● (black circle) indicates a bond to the portion on the T side, and O (white circle) indicates a bond to the portion on the B side. ] corresponding to any one chemical structure selected from the group consisting of any one of [71] to [83] or a salt thereof.
[85] The conjugate or salt thereof of any one of [71] to [84], wherein the functional substance is a drug or labeling substance.
[86] The complex or salt thereof according to any one of [71] to [85], wherein the functional substance is a low-molecular-weight compound.
[87] the conjugate or salt thereof of [85] or [86], wherein the drug is an anticancer agent;
[88] The complex or salt thereof of any one of [71] to [87], wherein the main chain connecting A and R' has 4 to 20 atoms.
[89] The complex or salt thereof of any one of [71] to [88], wherein the main chain connecting A and R does not contain a ring structure.
[90] The complex or salt thereof of any one of [71] to [89], wherein the partial structure represented by LB does not contain a peptide moiety.
[91] The compound represented by the formula (III) is represented by the following formula (III'):
A-B2'(-F2)-L'-B1'(-F1)-R'-T (III')
[In the formula,
A, R' and T are the same as in formula (II) above;
L' is a cleavable linker that is a divalent group containing a cleavable moiety;
B1' and B2' are the same or different and are divalent groups comprising moieties generated by reaction between a functional agent and a bioorthogonal functional group;
F1 and F2 are the same or different and are functional substances;
B1' (-F1) and B2' (-F2) may have a symmetrical structure about L'. ], the complex or a salt thereof according to any one of [71] to [90].
[92] The compound represented by the formula (III') is the following (III''):

[In the formula,
A, R' and T are the same as those of formula (III) described in [71],
C is a cleavable moiety;
p and p' are the same or different and any integer from 0 to 10;
q and q' are the same or different and any integer from 0 to 10;
X and X' are the same or different and are a carbon atom, a nitrogen atom, or a single bond (wherein when X is a nitrogen atom, R _1b is absent, and when X' is a nitrogen atom, R _{1b '} is absent.If X is a single bond, R _1a and R _1b are absent, and if X' is a single bond, R _1a' and R _1b' are absent);
R _1a , R _1b , R _1a′ and R _1b′ are the same or different and are selected from the group consisting of (i) to (vii);
Y and Y' are the same or different and are a residue obtained by removing one hydrogen atom from Y in formula (B-1) described in [32],
Z and Z' are the same or different and are the same as Z in the formula (B-1);
F and F' are the same or different and are functional substances. ], the complex of [91] or a salt thereof.
[93] the following formula (III):
ALB'(-F)-R'-T (III)
[In the formula,
A is an affinity substance for the antibody;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B' is a divalent group that includes a moiety generated by a reaction between a functional agent and a bioorthogonal functional group;
F is a functional substance,
R' is a moiety generated by reaction between an antibody and a reactive group specific for the side chain of a lysine residue;
T is an antibody. ], a complex having an affinity substance, a functional substance and an antibody or a salt thereof.

(Method for producing complex or salt thereof having affinity substance for soluble protein, cleavable moiety, functional substance and soluble protein)
[94] A method for producing a complex or a salt thereof comprising an affinity substance for a soluble protein, a cleavable moiety, a functional substance and a soluble protein,
Formula (II) below:
A-LB-R'-T (II)
[In the formula,
A is an affinity substance for soluble proteins;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B is (a) a divalent group comprising a bioorthogonal functional group;
R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein. ] and a soluble protein having a cleavable moiety or a salt thereof with a functional substance,
Formula (III) below:
ALB'(-F)-R'-T (III)
[In the formula,
A, L, R' and T are the same as in formula (II) above;
B' is a divalent group that includes a moiety generated by a reaction between a functional agent and a bioorthogonal functional group;
F is a functional substance. ], a conjugate having an affinity agent for a soluble protein, a cleavable moiety, a functional agent and a soluble protein, or a salt thereof.
[95] The method of [94], wherein the soluble protein is an antibody, and the reactive group is a reactive group specific to the side chain of a lysine residue.
[96] A method for producing a complex or a salt thereof comprising an affinity substance for a soluble protein, a cleavable moiety, a functional substance and a soluble protein,
(A) the following formula (I):
A-L-B-R (I)
[In the formula,
A is an affinity substance for soluble proteins;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B is (a) a divalent group comprising a bioorthogonal functional group;
R is a reactive group for said soluble protein. ], the compound or its salt is reacted with a soluble protein,
Formula (II) below:
A-LB-R'-T (II)
[In the formula,
A, L, and B are the same as in formula (I) above R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein. and (B) an affinity substance for said soluble protein and a soluble protein having a cleavable moiety or reacting the salt with a functional substance,
Formula (III) below:
ALB'(-F)-R'-T (III)
[In the formula,
A and L are the same as in formula (I) above;
R' and T are the same as in formula (II) above;
B' is a divalent group comprising a moiety generated by a reaction between a functional agent and a bioorthogonal functional group;
F is a functional substance. ], a conjugate having an affinity agent for a soluble protein, a cleavable moiety, a functional agent and a soluble protein, or a salt thereof.

Fourth, the present invention provides methods for producing soluble proteins with bioorthogonal functional groups.
[97] A method for producing a soluble protein having a bioorthogonal functional group or a salt thereof, comprising:
Formula (II) below:
A-LB-R'-T (II)
[In the formula,
A is an affinity substance for soluble proteins;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B is (a) a divalent group that includes a bioorthogonal functional group or (a) a divalent group that does not include a bioorthogonal functional group;
R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein. ], cleaving the cleavable portion of a soluble protein having an affinity for soluble proteins and a soluble protein having a cleavable portion or a salt thereof,
Formula (IV) below:
L1-B-R'-T (IV)
[In the formula,
B, R' and T are the same as in formula (II) above;
L1 is (i') a monovalent group that contains a bioorthogonal functional group or (ii') a monovalent group that does not contain a bioorthogonal functional group. ] or a salt thereof having a bioorthogonal functional group.
[98] L is (i) a cleavable linker that is a divalent group comprising a cleavable moiety capable of generating a bioorthogonal functional group on the side of the reactive group upon cleavage, or (ii) a bioorthogonal The method of [97], wherein the cleavable linker is a divalent group containing a cleavable moiety that does not have the ability to generate a functional group on the side of the reactive group.
[99] L is the cleavable linker of (i) above;
L1 is (i′) a monovalent group containing a bioorthogonal functional group;
The method of [98], wherein B is the divalent group of (a) or (b).
[100] L is the cleavable linker of (i);
L1 is (i′) a monovalent group containing a bioorthogonal functional group;
The method of [98] or [99], wherein B is the divalent group of (b).
[101] L is the cleavable linker of (ii);
L1 is (i′) a monovalent group that does not contain a bioorthogonal functional group;
The method of [98], wherein B is the divalent group of (a) above.
[102] the method of any one of [97] to [101], wherein the soluble protein is an antibody, and the reactive group is a reactive group specific to the side chain of a lysine residue;
[103] A method for producing a soluble protein having a bioorthogonal functional group or a salt thereof, comprising:
(A) the following formula (I):
A-L-B-R (I)
[In the formula,
A is an affinity substance for soluble proteins;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B is (a) a divalent group that includes a bioorthogonal functional group or (a) a divalent group that does not include a bioorthogonal functional group;
R is a reactive group for said soluble protein. ], the compound or its salt is reacted with a soluble protein,
Formula (II) below:
A-LB-R'-T (II)
[In the formula,
A, L, and B are the same as in formula (I) above R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein. and (B) an affinity substance for said soluble protein and a soluble protein having a cleavable moiety or cutting the cleavable portion of the salt,
Formula (IV) below:
L1-B-R'-T (IV)
[In the formula,
B, R' and T are the same as in formula (II) above;
L1 is (i') a monovalent group that contains a bioorthogonal functional group or (ii') a monovalent group that does not contain a bioorthogonal functional group. ] or a salt thereof having a bioorthogonal functional group.

Fifthly, the present invention provides a method for producing a soluble protein having a functional substance or a salt thereof.
[104] A method for producing a soluble protein having a functional substance or a salt thereof,
Formula (III) below:
ALB'(-F)-R'-T (III)
[In the formula,
A is an affinity substance for soluble proteins;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B' is a divalent group that includes a moiety generated by a reaction between a functional agent and a bioorthogonal functional group;
F is a functional substance,
R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein. ], or cleaving the cleavable portion of a complex having an affinity substance for a soluble protein, a cleavable portion, a functional substance and a soluble protein or a salt thereof, or the following formula (IV):
L1-B-R'-T (IV)
[In the formula,
L1 is (i′) a monovalent group containing a bioorthogonal functional group or (ii′) a monovalent group not containing a bioorthogonal functional group;
B is (a) a divalent group that includes a bioorthogonal functional group or (b) a divalent group that does not include a bioorthogonal functional group;
R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein. ], by reacting a soluble protein having a bioorthogonal functional group or a salt thereof with a functional substance,
Formula (V) below:
F-(L1-B)'-R'-T (V)
[In the formula,
L1 is (i′) a monovalent group containing a bioorthogonal functional group or (ii′) a monovalent group not containing a bioorthogonal functional group;
B is (a) a divalent group that includes a bioorthogonal functional group or (b) a divalent group that does not include a bioorthogonal functional group;
The structural unit represented by (L1-B)' includes a moiety generated by a reaction between a functional substance and either one or both of the bioorthogonal functional groups in (i') and (a). is a divalent structural unit,
F is a functional substance,
R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein. ], comprising producing a soluble protein having a functional substance or a salt thereof.
[105] cleaving the cleavable portion of the complex having the affinity substance for the soluble protein, the cleavable portion, the functional substance and the soluble protein or a salt thereof,
The following formula (V1):
L1-B'(-F)-R'-T (V1)
[In the formula,
L1 is (i′) a monovalent group containing a bioorthogonal functional group or (ii′) a monovalent group not containing a bioorthogonal functional group;
B', F, R' and T are the same as in formula (III) above. ], which comprises producing a soluble protein having a functional substance or a salt thereof.
[106] reacting the soluble protein having a bioorthogonal functional group or a salt thereof with one or two functional substances,
The following formula (V2):
FL1'-B-R'-T (V2)
[In the formula,
B, R' and T are the same as in formula (IV) above;
L1' is a divalent group comprising a moiety produced by a reaction between a functional agent and (i') a monovalent group comprising a bioorthogonal functional group;
F is a functional substance. ], or the following formula (V3):
Fa-L1'-B'(-Fb)-R'-T (V3)
[In the formula,
R' and T are the same as in formula (IV) above;
L1' is the same as in formula (V2) above,
B' is a divalent group that includes a moiety generated by a reaction between a functional agent and a bioorthogonal functional group;
Fa and Fb are the same or different functional substances, respectively. ], which comprises producing a soluble protein having a functional substance or a salt thereof.
[107] the method of any one of [104] to [106], wherein the soluble protein is an antibody and the reactive group is a reactive group specific to the side chain of a lysine residue;
[108] A method for producing a soluble protein having a functional substance or a salt thereof,
(A) the following formula (II):
A-LB-R'-T (II)
[In the formula,
A is an affinity substance for soluble proteins;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B is (a) a divalent group comprising a bioorthogonal functional group;
R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein. ] and a soluble protein having a cleavable moiety or a salt thereof with a functional substance,
Formula (III) below:
ALB'(-F)-R'-T (III)
[In the formula,
A, L, R' and T are the same as in formula (II) above;
B' is a divalent group that includes a moiety generated by a reaction between a functional agent and a bioorthogonal functional group;
F is a functional substance. ] and (B) the affinity substance for the soluble protein, the cleavable moiety , cleaving a cleavable portion of a complex having a functional agent and a soluble protein or a salt thereof,
The following formula (V1):
L1-B'(-F)-R'-T (V1)
[In the formula,
L1 is (i′) a monovalent group containing a bioorthogonal functional group or (ii′) a monovalent group not containing a bioorthogonal functional group;
B', F, R' and T are the same as in formula (III) above. ], comprising producing a soluble protein having a functional substance or a salt thereof.
[109] A method for producing a soluble protein having a functional substance or a salt thereof,
(A) the following formula (I):
A-L-B-R (I)
[In the formula,
A is an affinity substance for soluble proteins;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B is (a) a divalent group comprising a bioorthogonal functional group;
R is a reactive group for said soluble protein. ], a compound having an affinity for soluble proteins, a cleavable moiety and a reactive group or a salt thereof, is reacted with a soluble protein,
Formula (II) below:
A-LB-R'-T (II)
[In the formula,
A, L, and B are the same as in formula (I) above R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein. ], and producing a soluble protein or a salt thereof having a cleavable moiety,
(B) reacting an affinity substance for the soluble protein and a soluble protein having a cleavable moiety or a salt thereof with a functional substance,
Formula (III) below:
ALB'(-F)-R'-T (III)
[In the formula,
A and L are the same as in formula (I) above;
R' and T are the same as in formula (II) above;
B' is a divalent group that includes a moiety generated by a reaction between a functional agent and a bioorthogonal functional group;
F is a functional substance. ] and (C) the affinity substance for the soluble protein, the cleavable moiety , cleaving a cleavable portion of a complex having a functional agent and a soluble protein or a salt thereof,
The following formula (V1):
L1-B'(-F)-R'-T (V1)
[In the formula,
L1 is (i′) a monovalent group containing a bioorthogonal functional group or (ii′) a monovalent group not containing a bioorthogonal functional group;
B', F, R' and T are the same as in formula (III) above. ], comprising producing a soluble protein having a functional substance or a salt thereof.
[110] A method for producing a soluble protein having a functional substance or a salt thereof,
(A) the following formula (II):
A-LB-R'-T (II)
[In the formula,
A is an affinity substance for soluble proteins;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B is (a) a divalent group that includes a bioorthogonal functional group or (a) a divalent group that does not include a bioorthogonal functional group;
R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein. ], cleaving the cleavable portion of a soluble protein having an affinity for soluble proteins and a soluble protein having a cleavable portion or a salt thereof,
Formula (IV) below:
L1-B-R'-T (IV)
[In the formula,
B, R' and T are the same as in formula (II) above;
L1 is (i') a monovalent group that contains a bioorthogonal functional group or (ii') a monovalent group that does not contain a bioorthogonal functional group. ], and (B) the soluble protein having a bioorthogonal functional group or a salt thereof represented by one or more functions by reacting with a substance,
The following formula (V2)
FL1'-B-R'-T (V2)
[In the formula,
B, R' and T are the same as in formula (IV) above;
L1' is a divalent group comprising a moiety produced by a reaction between a functional agent and (i') a monovalent group comprising a bioorthogonal functional group;
F is a functional substance. ], or the following formula (V3):
Fa-L1'-B'(-Fb)-R'-T (V3)
[In the formula,
R' and T are the same as in formula (IV) above;
L1' is the same as in formula (V2) above,
B' is a divalent group that includes a moiety generated by a reaction between a functional agent and a bioorthogonal functional group;
Fa and Fb are the same or different functional substances, respectively. ], comprising producing a soluble protein having a functional substance or a salt thereof.
[111] A method for producing a soluble protein having a functional substance or a salt thereof,
(A) the following formula (I):
A-L-B-R (I)
[In the formula,
A is an affinity substance for soluble proteins;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B is (a) a divalent group that includes a bioorthogonal functional group or (a) a divalent group that does not include a bioorthogonal functional group;
R is a reactive group for said soluble protein. ], the compound or its salt is reacted with a soluble protein,
Formula (II) below:
A-LB-R'-T (II)
[In the formula,
A, L, and B are the same as in formula (I) above R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein. ], and producing a soluble protein or a salt thereof having a cleavable moiety,
(B) cleaving a cleavable portion of a soluble protein having an affinity for said soluble protein and a soluble protein having a cleavable portion or a salt thereof,
Formula (IV) below:
L1-B-R'-T (IV)
[In the formula,
B, R' and T are the same as in formula (II) above;
L1 is (i') a monovalent group that contains a bioorthogonal functional group or (ii') a monovalent group that does not contain a bioorthogonal functional group. and (C) the soluble protein having a bioorthogonal functional group or a salt thereof represented by one or more functions by reacting with a substance,
The following formula (V2)
FL1'-B-R'-T (V2)
[In the formula,
B, R' and T are the same as in formula (IV) above;
L1' is a divalent group comprising a moiety produced by a reaction between a functional agent and (i') a monovalent group comprising a bioorthogonal functional group;
F is a functional substance. ], or the following formula (V3):
Fa-L1'-B'(-Fb)-R'-T (V3)
[In the formula,
R' and T are the same as in formula (IV) above;
L1' is the same as in formula (V2) above,
B' is a divalent group that includes a moiety generated by a reaction between a functional agent and a bioorthogonal functional group;
Fa and Fb are the same or different functional substances, respectively. ], comprising producing a soluble protein having a functional substance or a salt thereof.

〔式中、
ｐは、０～１０の任意の整数であり、
ｑは、０～１０の任意の整数であり、
Ｘは、炭素原子、窒素原子、または単結合（ここで、Ｘが窒素原子である場合、Ｒ_１ｂは存在しない。Ｘが単結合である場合、Ｒ_１ａおよびＲ_１ｂは存在しない）であり、
Ｒ_１ａ、およびＲ_１ｂは、同一または異なって、水素原子、または上述する置換基からなる群より選ばれる。
○（白丸）は、Ｃ１に対する結合を示し、●（黒丸）はＢに対する結合を示す。〕で表される、〔１９〕の可溶性タンパク質またはその塩。
〔２１〕生体直交性官能基を含む２価の基が、アジド残基、アルデヒド残基、チオール残基、アルキン残基、アルケン残基、テトラジン残基、ニトロン残基、ヒドロキシルアミン残基、ニトリル残基、ヒドラジン残基、ケトン残基、ボロン酸残基、シアノベンゾチアゾール残基、アリル残基、ホスフィン残基、マレイミド残基、ジスルフィド残基、チオエステル基、α―ハロカルボニル残基、イソニトリル残基、シドノン残基、セレン残基からなる群より選ばれる生体直交性官能基を主鎖に含む２価の基である、〔１〕～〔２０〕のいずれかの可溶性タンパク質またはその塩。
〔２２〕生体直交性官能基を含む２価の基が、アジド残基、アルデヒド残基、チオール残基、アルキン残基、アルケン残基、ハロゲン残基、テトラジン残基、ニトロン残基、ヒドロキシルアミン残基、ニトリル残基、ヒドラジン残基、ケトン残基、ボロン酸残基、シアノベンゾチアゾール残基、アリル残基、ホスフィン残基、マレイミド残基、ジスルフィド残基、α―ハロカルボニル残基、イソニトリル残基、シドノン残基、セレン残基からなる群より選ばれる生体直交性官能基を側鎖に含む２価の基である、〔１〕～〔２０〕のいずれかの可溶性タンパク質またはその塩。
〔２３〕生体直交性官能基が、下記：

〔式中、
Ｒ_１ｆ、単一もしくは複数のＲ_１ｇおよび単一もしくは複数のＲ_１ｈは、同一もしくは異なって、前記（ｉ）～（ｖｉｉ）からなる群より選ばれる原子もしくは基、または電子吸引基であり、
・は、結合手である。）で表されるいずれか一つである、〔１〕～〔２２〕のいずれかの可溶性タンパク質またはその塩。
〔２４〕前記（ｂ）の２価の基が、置換されていてもよいアルキレン、置換されていてもよいシクロアルキレン、置換されていてもよいアリール、置換されていてもよい２価の複素環基、－ＮＲ_ａ－（Ｒ_ａは水素原子、または置換基を示す）、－Ｏ－、またはこれらの２以上の組み合わせからなる群より選ばれる、〔２〕～〔１３〕、〔１６〕～〔２３〕のいずれかの化合物またはその塩。
〔２５〕Ｂが、下記式（Ｂ－１）：

〔式中、
Ｙは、－ＮＨ－、－Ｏ－、－ＣＨ_２－、または下記式（Ｂ－２）：

（式中、
ＶおよびＶ’は、同一または異なって、－ＮＨ－、－Ｏ－、－ＣＨ_２－、または単結合であり、
Ｖ１は、生体直交性官能基を含む２価の基であり、
ｓは、０～１０の任意の整数であり、
式（Ｂ－２）における○および●は、それぞれ、式（Ｂ－１）における○および●と同じ配向である。）であり、
Ｚは、酸素原子、硫黄原子、または水素原子（Ｚが水素原子である場合、－Ｃ（＝Ｚ）－は、－ＣＨ_２－を示す。）であり、
式（Ｂ－１）における○（白丸）は、Ｌ側の部分に対する結合を示し、●（黒丸）はＲ側の部分に対する結合を示す。〕で表される、〔２〕～〔１３〕、〔１６〕～〔２４〕のいずれかの可溶性タンパク質またはその塩。
〔２６〕生体直交性官能基が、リジン残基、チロシン残基、またはトリプトファン残基のいずれか一つの側鎖を介して可溶性タンパク質に結合している、〔１〕～〔２５〕のいずれかの可溶性タンパク質またはその塩。
〔２７〕生体直交性官能基が、リジン残基の側鎖を介して可溶性タンパク質に結合している、〔２６〕の可溶性タンパク質またはその塩。
〔２８〕反応性基が、リジン残基、チロシン残基、またはトリプトファン残基のいずれか一つの側鎖に対して特異的な反応性基である、〔２〕～〔１３〕、〔１６〕～〔２６〕のいずれかの可溶性タンパク質またはその塩。
〔２９〕反応性基が、リジン残基の側鎖に対して特異的な反応性基である、〔２〕～〔１３〕、〔１６〕～〔２８〕のいずれかの可溶性タンパク質またはその塩。
〔３０〕反応性基が、下記：

〔ここで、
Ｒ_５ａおよびＲ_５cは、前記（ｉ）～（ｖｉｉ）からなる群より選ばれる原子または基であり、
Ｒ_５ｂは、電子吸引基であり、
ｊは、１～５の任意の整数であり、
ｋは、１～４の任意の整数である。〕からなる群より選ばれるいずれか一つの化学構造に対応する、〔２〕～〔１３〕、〔１６〕～〔２９〕のいずれかの可溶性タンパク質またはその塩。
〔３１〕生体直交性官能基および特定のアミノ酸残基の側鎖を連結する主鎖の原子数が２～１０個であるリンカーを介して生体直交性官能基が可溶性タンパク質に結合している、〔１〕～〔３０〕のいずれかの可溶性タンパク質またはその塩。
〔３２〕生体直交性官能基および特定のアミノ酸残基の側鎖を連結する主鎖が環構造を含まないリンカーを介して生体直交性官能基が可溶性タンパク質に結合している、〔１〕～〔３１〕のいずれかの可溶性タンパク質またはその塩。
〔３３〕Ｌ１末端部分およびＲ’を連結する主鎖の原子数が２～１０個である、〔２〕～〔１３〕、〔１６〕～〔３２〕のいずれかの可溶性タンパク質またはその塩。
〔３４〕ＡおよびＲを連結する主鎖が環構造を含まない、〔２〕～〔１３〕、〔１６〕～〔３３〕のいずれかの可溶性タンパク質またはその塩。
〔３５〕Ｌ１－Ｂで表される部分構造がペプチド部分を含まない、〔２〕～〔１３〕、〔１６〕～〔３４〕のいずれかの可溶性タンパク質またはその塩。
〔３６〕前記式（ＩＶ）で表される可溶性タンパク質が、下記（ＩＶ’）：

〔式中、
Ｃ１は、生体直交性官能基、または生体直交性官能基以外の基である。
ｐ、ｑ、Ｘ、Ｒ_１ａおよびＲ_１ｂは、前記式（Ｌｂ’）のものと同じであり、
ＹおよびＺは、前記のものと同じであり、
Ｒ’およびＴは、前記式（ＩＶ）のものと同じである。〕で表される、〔２〕～〔１３〕、〔１６〕～〔３５〕のいずれかの可溶性タンパク質またはその塩。
〔３７〕生体直交性官能基を位置選択的に有する可溶性タンパク質またはその塩であって
、
可溶性タンパク質が、連続する１～５０個のアミノ酸残基からなる標的領域中において特定のアミノ酸残基を１個以上含み、かつ、前記標的領域以外の非標的領域中において前記特定のアミノ酸残基を５個以上含み、かつ
生体直交性官能基が、ペプチド部分を含まないリンカーを介して、前記標的領域中に含まれる１個以上の特定のアミノ酸残基に対して３０％以上の位置選択性で結合しており、
生体直交性官能基がリジン残基の側鎖を介して可溶性タンパク質に結合しており、
可溶性タンパク質が、複数の重鎖を含む抗体であり、
抗体に含まれる複数の重鎖中の前記位置に存在する複数の特定のアミノ酸残基において生体直交性官能基を有する結果、抗体が複数の生体直交性官能基を有する、生体直交性官能基を位置選択的に有する可溶性タンパク質またはその塩。
〔３８〕下記式（ＩＶ）：
Ｌ１－Ｂ－Ｒ’－Ｔ （ＩＶ）
〔式中、
Ｌ１は、（ｉ’）生体直交性官能基を含む１価の基、または（ｉｉ’）生体直交性官能基を含まない１価の基であり、
Ｂは、（ａ）生体直交性官能基を含む２価の基、または（ｂ）生体直交性官能基を含まない２価の基であり、
Ｒ’は、抗体と、リジン残基の側鎖に対して特異的な反応性基との間の反応により生成する部分であり、
Ｔは、可溶性タンパク質であり、
前記可溶性タンパク質は、連続する１～５０個のアミノ酸残基からなる標的領域中において特定のアミノ酸残基を１個以上含み、かつ、前記標的領域以外の非標的領域中において前記特定のアミノ酸残基を５個以上含む。〕で表され、かつＬ１－Ｂ－Ｒ’で表される構造単位が、前記可溶性タンパク質の標的領域中に含まれる１個以上の特定のアミノ酸残基に対して３０％以上の位置選択性で結合している、生体直交性官能基を位置選択的に有する可溶性タンパク質またはその塩。 Sixthly, the present invention provides a soluble protein or a salt thereof regioselectively having a bioorthogonal functional group, and a method for producing the same.
(Soluble protein or salt thereof regioselectively having a bioorthogonal functional group)
[1] A soluble protein or a salt thereof regioselectively having a bioorthogonal functional group,
The soluble protein contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and the specific amino acid residue in a non-target region other than the target region 5 or more bioorthogonal functional groups with a regioselectivity of 30% or more for one or more specific amino acid residues contained in the target region via a linker that does not contain a peptide moiety A soluble protein or salt thereof having a regioselectively attached bioorthogonal functional group.
[2] Formula (IV) below:
L1-B-R'-T (IV)
[In the formula,
L1 is (i′) a monovalent group containing a bioorthogonal functional group or (ii′) a monovalent group not containing a bioorthogonal functional group;
B is (a) a divalent group that includes a bioorthogonal functional group or (b) a divalent group that does not include a bioorthogonal functional group;
R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein,
The soluble protein contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and the specific amino acid residue in a non-target region other than the target region 5 or more. ] and the structural unit represented by L1-BR' has a regioselectivity of 30% or more with respect to one or more specific amino acid residues contained in the target region of the soluble protein A soluble protein or salt thereof having a regioselectively attached bioorthogonal functional group.
[3] The soluble protein of [1] or [2] or a salt thereof, wherein the soluble protein is a monoclonal antibody.
[4] The soluble protein of [1] to [3] or a salt thereof, wherein the soluble protein is an IgG antibody.
[5] The soluble protein or salt thereof according to any one of [1] to [4], wherein the soluble protein is of human origin.
[6] any one of [1] to [5], wherein the soluble protein comprises any one Fc region protein selected from the group consisting of the following (A) to (C) and is an antibody having antigen-binding ability: Soluble protein or salt thereof:
(A) an Fc region protein comprising the amino acid sequence of SEQ ID NO: 1;
(B) an Fc region protein comprising an amino acid sequence in which one or several amino acid residues are inserted, added, deleted or substituted in the amino acid sequence of SEQ ID NO: 1; or (C) the amino acid sequence of SEQ ID NO: 1 and 90 An Fc region protein comprising an amino acid sequence exhibiting greater than or equal to % identity.
[7] The soluble protein or salt thereof according to any one of [1] to [6], wherein the target region is a region consisting of 1 to 10 consecutive amino acid residues.
[8] The soluble protein or salt thereof according to any one of [1] to [7], wherein the target region is a region consisting of 1 to 3 consecutive amino acid residues.
[9] the target region is (a) a region consisting of amino acid residues at positions 246-248 in the human IgG Fc region, (b) a region consisting of amino acid residues at positions 288-290 in the human IgG Fc region, or ( c) The soluble protein of [8], which is a region consisting of amino acid residues at position 317 in the human IgG Fc region, or a salt thereof.
[10] The soluble protein or salt thereof according to any one of [1] to [9], wherein the regioselectivity is 50% or more.
[11] The soluble protein or salt thereof of [10], wherein the regioselectivity is 70% or more.
[12] The soluble protein or salt thereof of [11], wherein the regioselectivity is 90% or more.
[13] the specific amino acid residue is a (where a is an arbitrary integer of 1 to 10 ) does not contain amino acid residues of the same type as the specific amino acid residue other than the specific amino acid residue present at the specific position in the region up to the remote position of the amino acid residue of [1] to [ 12] any soluble protein or a salt thereof.
[14] the soluble protein is a multimeric protein comprising a plurality of monomeric proteins,
Having a bioorthogonal functional group at a plurality of specific amino acid residues present at the positions in a plurality of monomeric proteins contained in the multimeric protein results in the multimeric protein having a plurality of bioorthogonal functional groups. , [1], [3] to [13] or a soluble protein or a salt thereof.
[15] the soluble protein is an antibody comprising a plurality of heavy chains;
[1], [3], wherein the antibody has a plurality of bioorthogonal functional groups as a result of having bioorthogonal functional groups at a plurality of specific amino acid residues present at the positions in the plurality of heavy chains contained in the antibody; ] to [14] any soluble protein or a salt thereof.
[16] the soluble protein is a multimeric protein comprising a plurality of monomeric proteins,
T has a structural unit represented by ALBR' in a plurality of corresponding target regions in a plurality of monomeric proteins, resulting in a multimeric protein represented by ALBR' A soluble protein or a salt thereof according to any one of [2] to [13], having a plurality of structural units represented.
[17] the soluble protein is an antibody comprising a plurality of heavy chains;
T has structural units represented by ALBR' in corresponding multiple target regions in multiple heavy chains, resulting in an antibody having a structure represented by ALBR' A soluble protein or a salt thereof according to any one of [2] to [13] and [16], which has multiple units.
[18] The soluble protein of [15] or [17], which has two heavy chains, or a salt thereof.
[19] L1 is represented by the following formulas (L1-1) to (L1-2):
C1-Lb (L1-1)
C1 (L1-2)
[In the formula,
Lb is a divalent group,
C1 is a bioorthogonal functional group or a group other than a bioorthogonal functional group. ], the soluble protein of any one of [2] to [13] and [16] to [18] or a salt thereof.
[20] Lb is represented by the following formula (Lb'):

[In the formula,
p is any integer from 0 to 10,
q is any integer from 0 to 10,
X is a carbon atom, a nitrogen atom, or a single bond (wherein when X is a nitrogen atom, R _1b is absent; when X is a single bond, R _1a and R _1b are absent);
R _1a and R _1b are the same or different and are selected from the group consisting of a hydrogen atom or the substituents described above.
O (white circle) indicates binding to C1 and ● (filled circle) indicates binding to B. ], the soluble protein of [19] or a salt thereof.
[21] the bivalent group containing a bioorthogonal functional group is an azide residue, an aldehyde residue, a thiol residue, an alkyne residue, an alkene residue, a tetrazine residue, a nitrone residue, a hydroxylamine residue, a nitrile residue, hydrazine residue, ketone residue, boronic acid residue, cyanobenzothiazole residue, allyl residue, phosphine residue, maleimide residue, disulfide residue, thioester group, α-halocarbonyl residue, isonitrile residue The soluble protein or salt thereof according to any one of [1] to [20], which is a divalent group containing a bioorthogonal functional group selected from the group consisting of groups, sydnon residues and selenium residues in its main chain.
[22] the bivalent group containing a bioorthogonal functional group is an azide residue, an aldehyde residue, a thiol residue, an alkyne residue, an alkene residue, a halogen residue, a tetrazine residue, a nitrone residue, hydroxylamine; residue, nitrile residue, hydrazine residue, ketone residue, boronic acid residue, cyanobenzothiazole residue, allyl residue, phosphine residue, maleimide residue, disulfide residue, α-halocarbonyl residue, isonitrile The soluble protein or salt thereof according to any one of [1] to [20], which is a divalent group containing a bioorthogonal functional group selected from the group consisting of residues, sydnon residues and selenium residues in its side chain.
[23] the bioorthogonal functional group is:

[In the formula,
R _1f , single or multiple R _1g and single or multiple R _1h are the same or different and are atoms or groups selected from the group consisting of (i) to (vii), or electron-withdrawing groups;
・ is a bond. ), the soluble protein or a salt thereof according to any one of [1] to [22].
[24] The divalent group (b) is optionally substituted alkylene, optionally substituted cycloalkylene, optionally substituted aryl, or optionally substituted bivalent heterocyclic ring group, -NR _a - (R _a represents a hydrogen atom or a substituent), -O-, or selected from the group consisting of a combination of two or more thereof [2] to [13], [16] to The compound of [23] or a salt thereof.
[25] B is the following formula (B-1):

[In the formula,
Y is -NH-, -O-, -CH ₂ -, or the following formula (B-2):

(In the formula,
V and V′ are the same or different and are —NH—, —O—, —CH ₂ —, or a single bond;
V is a divalent group containing a bioorthogonal functional group;
s is any integer from 0 to 10,
○ and ● in formula (B-2) have the same orientations as ○ and ● in formula (B-1), respectively. ) and
Z is an oxygen atom, a sulfur atom, or a hydrogen atom (when Z is a hydrogen atom, -C(=Z)- represents -CH ₂ -);
○ (white circle) in formula (B-1) indicates a bond to the L-side portion, and ● (black circle) indicates a bond to the R-side portion. ], a soluble protein or a salt thereof according to any one of [2] to [13] and [16] to [24].
[26] any one of [1] to [25], wherein the bioorthogonal functional group is bound to a soluble protein via a side chain of any one of a lysine residue, a tyrosine residue, or a tryptophan residue; soluble protein or a salt thereof.
[27] The soluble protein of [26] or a salt thereof, wherein the bioorthogonal functional group is bound to the soluble protein via a side chain of a lysine residue.
[28] the reactive group is a reactive group specific to any one side chain of a lysine residue, a tyrosine residue, or a tryptophan residue, [2] to [13], [16] The soluble protein or salt thereof according to any one of ~[26].
[29] The soluble protein or salt thereof of any one of [2] to [13] and [16] to [28], wherein the reactive group is a reactive group specific to the side chain of a lysine residue .
[30] the reactive group is:

〔here,
R _5a and R _5c are atoms or groups selected from the group consisting of (i) to (vii);
R _5b is an electron withdrawing group,
j is any integer from 1 to 5;
k is any integer from 1 to 4. A soluble protein or a salt thereof according to any one of [2] to [13] and [16] to [29] corresponding to any one chemical structure selected from the group consisting of ].
[31] the bioorthogonal functional group is bound to the soluble protein via a linker having a main chain of 2 to 10 atoms that connects the bioorthogonal functional group and the side chain of a specific amino acid residue; A soluble protein or a salt thereof according to any one of [1] to [30].
[32] The bioorthogonal functional group is bound to a soluble protein via a linker in which the main chain connecting the bioorthogonal functional group and the side chain of a specific amino acid residue does not contain a ring structure, [1]- [31] Any soluble protein or salt thereof.
[33] The soluble protein or salt thereof according to any one of [2] to [13] and [16] to [32], wherein the main chain connecting the L1 terminal portion and R' has 2 to 10 atoms.
[34] The soluble protein or salt thereof according to any one of [2] to [13] and [16] to [33], wherein the main chain connecting A and R does not contain a ring structure.
[35] The soluble protein or salt thereof according to any one of [2] to [13] and [16] to [34], wherein the partial structure represented by L1-B does not contain a peptide moiety.
[36] The soluble protein represented by the formula (IV) is the following (IV'):

[In the formula,
C1 is a bioorthogonal functional group or a group other than a bioorthogonal functional group.
p, q, X, R _1a and R _1b are the same as in formula (Lb′) above;
Y and Z are the same as above;
R' and T are the same as in formula (IV) above. ], a soluble protein or a salt thereof according to any one of [2] to [13] and [16] to [35].
[37] A soluble protein or a salt thereof regioselectively having a bioorthogonal functional group,
The soluble protein contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and the specific amino acid residue in a non-target region other than the target region 5 or more bioorthogonal functional groups with a regioselectivity of 30% or more for one or more specific amino acid residues contained in the target region via a linker that does not contain a peptide moiety is bound,
bioorthogonal functional groups are attached to soluble proteins through the side chains of lysine residues,
the soluble protein is an antibody comprising multiple heavy chains;
A bioorthogonal functional group in which the antibody has a plurality of bioorthogonal functional groups as a result of having a bioorthogonal functional group at a plurality of specific amino acid residues present at the positions in the plurality of heavy chains contained in the antibody. A soluble protein having regioselectivity or a salt thereof.
[38] Formula (IV) below:
L1-B-R'-T (IV)
[In the formula,
L1 is (i′) a monovalent group containing a bioorthogonal functional group or (ii′) a monovalent group not containing a bioorthogonal functional group;
B is (a) a divalent group that includes a bioorthogonal functional group or (b) a divalent group that does not include a bioorthogonal functional group;
R' is a moiety generated by reaction between an antibody and a reactive group specific for the side chain of a lysine residue;
T is a soluble protein,
The soluble protein contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and the specific amino acid residue in a non-target region other than the target region 5 or more. ] and the structural unit represented by L1-BR' has a regioselectivity of 30% or more with respect to one or more specific amino acid residues contained in the target region of the soluble protein A soluble protein or salt thereof having a regioselectively attached bioorthogonal functional group.

(Method for producing soluble protein or salt thereof regioselectively having bioorthogonal functional group)
The present invention also provides a soluble protein or a salt thereof regioselectively having a bioorthogonal functional group, which requires specification by formula (IV) or subordinate formulas thereof, among the soluble proteins [1] to [38] above. A manufacturing method is provided.
[39] A method for producing a soluble protein or a salt thereof regioselectively having a bioorthogonal functional group, comprising:
Formula (II) below:
A-LB-R'-T (II)
[In the formula,
A is an affinity substance for soluble proteins;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B is (a) a divalent group that includes a bioorthogonal functional group or (a) a divalent group that does not include a bioorthogonal functional group;
R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein,
The soluble protein contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and the specific amino acid residue in a non-target region other than the target region 5 or more. ] and the structural unit represented by L1-BR' has a regioselectivity of 30% or more with respect to one or more specific amino acid residues contained in the target region of the soluble protein cleaving the bound affinity substance for the soluble protein and the cleavable portion of the soluble protein or salt thereof regioselectively carrying the cleavable portion,
Formula (IV) below:
L1-B-R'-T (IV)
[In the formula,
B, R' and T are the same as in formula (II) above;
L1 is (i') a monovalent group that contains a bioorthogonal functional group or (ii') a monovalent group that does not contain a bioorthogonal functional group. ] and the structural unit represented by L1-BR' has a regioselectivity of 30% or more with respect to one or more specific amino acid residues contained in the target region of the soluble protein. A method comprising producing a soluble protein or salt thereof regioselectively having an attached bioorthogonal functional group.

Preferably, the method of [39] above may be as follows.
[40] The method of [39], wherein the soluble protein is an antibody, and the reactive group is a reactive group specific to the side chain of a lysine residue.
[41] A method for producing a soluble protein or a salt thereof regioselectively having a bioorthogonal functional group, comprising:
(A) the following formula (I):
A-L-B-R (I)
[In the formula,
A is an affinity substance for soluble proteins;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B is (a) a divalent group that includes a bioorthogonal functional group or (a) a divalent group that does not include a bioorthogonal functional group;
R is a reactive group for said soluble protein. ] A compound or a salt thereof represented by a soluble protein (here, the soluble protein contains one or more specific amino acid residues in the target region consisting of 1 to 50 consecutive amino acid residues, and containing 5 or more of the specific amino acid residues in a non-target region other than the target region),
Formula (II) below:
A-LB-R'-T (II)
[In the formula,
A, L, and B are the same as in formula (I) above R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein. ] and the structural unit represented by L1-BR' has a regioselectivity of 30% or more with respect to one or more specific amino acid residues contained in the target region of the soluble protein Generating a soluble protein or a salt thereof regioselectively having an affinity substance for a soluble protein and a cleavable moiety attached thereto, and (B) having an affinity substance for said soluble protein and a cleavable moiety cleaving a cleavable portion of the soluble protein or salt thereof,
Formula (IV) below:
L1-B-R'-T (IV)
[In the formula,
B, R' and T are the same as in formula (II) above;
L1 is (i') a monovalent group that contains a bioorthogonal functional group or (ii') a monovalent group that does not contain a bioorthogonal functional group. ] and the structural unit represented by L1-BR' has a regioselectivity of 30% or more with respect to one or more specific amino acid residues contained in the target region of the soluble protein A method comprising producing a soluble protein or salt thereof regioselectively having an attached bioorthogonal functional group.

〔式中、
ｐは、０～１０の任意の整数であり、
ｑは、０～１０の任意の整数であり、
Ｘは、炭素原子、窒素原子、または単結合（ここで、Ｘが窒素原子である場合、Ｒ_１ｂは存在しない。Ｘが単結合である場合、Ｒ_１ａおよびＲ_１ｂは存在しない）であり、
Ｒ_１ａ、およびＲ_１ｂは、同一または異なって、水素原子、または上述する置換基からなる群より選ばれる。
○（白丸）は、Ｃ１に対する結合を示し、●（黒丸）はＢに対する結合を示す。〕で表される、〔２１〕の可溶性タンパク質またはその塩。
〔２３〕生体直交性官能基を含む２価の基が、アジド残基、アルデヒド残基、チオール残基、アルキン残基、アルケン残基、テトラジン残基、ニトロン残基、ヒドロキシルアミン残基、ニトリル残基、ヒドラジン残基、ケトン残基、ボロン酸残基、シアノベンゾチアゾール残基、アリル残基、ホスフィン残基、マレイミド残基、ジスルフィド残基、チオエステル基、α―ハロカルボニル残基、イソニトリル残基、シドノン残基、セレン残基からなる群より選ばれる生体直交性官能基を主鎖に含む２価の基である、〔１〕～〔２２〕のいずれかの可溶性タンパク質またはその塩。
〔２４〕生体直交性官能基を含む２価の基が、アジド残基、アルデヒド残基、チオール残基、アルキン残基、アルケン残基、ハロゲン残基、テトラジン残基、ニトロン残基、ヒドロキシルアミン残基、ニトリル残基、ヒドラジン残基、ケトン残基、ボロン酸残基、シアノベンゾチアゾール残基、アリル残基、ホスフィン残基、マレイミド残基、ジスルフィド残基、α―ハロカルボニル残基、イソニトリル残基、シドノン残基、セレン残基からなる
群より選ばれる生体直交性官能基を側鎖に含む２価の基である、〔１〕～〔２２〕のいずれかの可溶性タンパク質またはその塩。
〔２５〕生体直交性官能基が、下記：

〔式中、
Ｒ_１ｆ、単一もしくは複数のＲ_１ｇおよび単一もしくは複数のＲ_１ｈは、同一もしくは異なって、前記（ｉ）～（ｖｉｉ）からなる群より選ばれる原子もしくは基、または電子吸引基であり、
・は、結合手である。）で表されるいずれか一つである、〔１〕～〔２４〕のいずれかの可溶性タンパク質またはその塩。
〔２６〕前記（ｂ）の２価の基が、置換されていてもよいアルキレン、置換されていてもよいシクロアルキレン、置換されていてもよいアリール、置換されていてもよい２価の複素環基、－ＮＲ_ａ－（Ｒ_ａは水素原子、または置換基を示す）、－Ｏ－、またはこれらの２以上の組み合わせからなる群より選ばれる、〔１〕～〔２５〕のいずれかの可溶性タンパク質またはその塩。
〔２７〕Ｂが、下記式（Ｂ－１）：

〔式中、
Ｙは、－ＮＨ－、－Ｏ－、－ＣＨ_２－、または下記式（Ｂ－２）：

（式中、
ＶおよびＶ’は、同一または異なって、－ＮＨ－、－Ｏ－、－ＣＨ_２－、または単結合であり、
Ｖ１は、生体直交性官能基を含む２価の基であり、
ｓは、０～１０の任意の整数であり、
式（Ｂ－２）における○および●は、それぞれ、式（Ｂ－１）における○および●と同じ配向である。）であり、
Ｚは、酸素原子、硫黄原子、または水素原子（Ｚが水素原子である場合、－Ｃ（＝Ｚ）－は、－ＣＨ_２－を示す。）であり、
式（Ｂ－１）における○（白丸）は、Ｌ側の部分に対する結合を示し、●（黒丸）はＲ側の部分に対する結合を示す。〕で表される、〔２〕～〔１５〕、〔１８〕～〔２６〕のいずれかの可溶性タンパク質またはその塩。
〔２８〕反応性基が、リジン残基、チロシン残基、またはトリプトファン残基のいずれか一つの側鎖に対して特異的な反応性基である、〔２〕～〔１５〕、〔１８〕～〔２７〕のいずれかの可溶性タンパク質またはその塩。
〔２９〕反応性基が、リジン残基の側鎖に対して特異的な反応性基である、〔２〕～〔１５〕、〔１８〕～〔２８〕のいずれかの可溶性タンパク質またはその塩。
〔３０〕反応性基が、下記：

〔ここで、
Ｒ_５ａおよびＲ_５cは、前記（ｉ）～（ｖｉｉ）からなる群より選ばれる原子または基であり、
Ｒ_５ｂは、電子吸引基であり、
ｊは、１～５の任意の整数であり、
ｋは、１～４の任意の整数である。〕からなる群より選ばれるいずれか一つの化学構造に対応する、〔２〕～〔１５〕、〔１８〕～〔２９〕のいずれかの可溶性タンパク質またはその塩。
〔３１〕機能性物質および特定のアミノ酸残基の側鎖を連結する主鎖の原子数が２～１０個であるリンカーを介して生体直交性官能基が可溶性タンパク質に結合している、〔１〕～〔３０〕のいずれかの可溶性タンパク質またはその塩。
〔３２〕機能性物質および特定のアミノ酸残基の側鎖を連結する主鎖が環構造を含まないリンカーを介して生体直交性官能基が可溶性タンパク質に結合している、〔１〕～〔３１〕のいずれかの可溶性タンパク質またはその塩。
〔３３〕Ｌ１末端部分およびＲ’を連結する主鎖の原子数が２～１０個である、〔２〕～〔１５〕、〔１８〕～〔３２〕のいずれかの可溶性タンパク質またはその塩。
〔３４〕ＡおよびＲを連結する主鎖が環構造を含まない、〔２〕～〔１５〕、〔１８〕～〔３３〕のいずれかの可溶性タンパク質またはその塩。
〔３５〕Ｌ１－Ｂで表される部分構造がペプチド部分を含まない、〔２〕～〔１５〕、〔
１８〕～〔３４〕のいずれかの可溶性タンパク質またはその塩。
〔３６〕前記式（Ｖ１）、（Ｖ２）および（Ｖ３）で表される、機能性物質を位置選択的に有する可溶性タンパク質またはその塩が、それぞれ、下記（Ｖ１’）、（Ｖ２’）および（Ｖ３’）：

〔式中、
Ｃ１は、生体直交性官能基、または生体直交性官能基以外の基である。
ｐ、ｑ、Ｘ、Ｒ_１ａおよびＲ_１ｂは、前記式（Ｌｂ’）のものと同じであり、
Ｙ’は、前記式（Ｂ－１）のＹから水素原子を一つ除いた残基であり、
Ｚは、前記式（Ｂ－１）のものと同じであり、
Ｆ、Ｒ’およびＴは、前記式（Ｖ）のものと同じである。〕、

〔式中、
Ｃ１’は、機能性物質と生体直交性官能基との間の反応により生成する部分である。
ｐ、ｑ、Ｘ、Ｒ_１ａおよびＲ_１ｂは、前記式（Ｌｂ’）のものと同じであり、
ＹおよびＺは、前記式（Ｂ－１）のものと同じであり、
Ｆ、Ｒ’およびＴは、前記式（Ｖ）のものと同じである。〕、あるいは

〔式中、
Ｃ１’は、機能性物質と生体直交性官能基との間の反応により生成する部分である。
ｐ、ｑ、Ｘ、Ｒ_１ａおよびＲ_１ｂは、前記式（Ｌｂ’）のものと同じであり、
Ｙ’は、前記式（Ｂ－１）のＹから水素原子を一つ除いた残基であり、
Ｚは、前記式（Ｂ－１）のものと同じであり、
ＦａおよびＦｂは、同一または異なる機能性物質であり、
Ｒ’およびＴは、前記式（Ｖ）のものと同じである。〕で表される、機能性物質を位置選択的に有する可溶性タンパク質またはその塩である、〔２〕～〔１５〕、〔１８〕～〔３５〕のいずれかの可溶性タンパク質またはその塩。
〔３７〕機能性物質を位置選択的に有する可溶性タンパク質またはその塩であって、
可溶性タンパク質が、連続する１～５０個のアミノ酸残基からなる標的領域中において特定のアミノ酸残基を１個以上含み、かつ、前記標的領域以外の非標的領域中において前記特定のアミノ酸残基を５個以上含み、かつ
機能性物質が、ペプチド部分を含まないリンカーを介して、前記標的領域中に含まれる１個以上の特定のアミノ酸残基に対して３０％以上の位置選択性で結合しており、
機能性物質がリジン残基の側鎖を介して可溶性タンパク質に結合しており、
可溶性タンパク質が、複数の重鎖を含む抗体であり、
抗体に含まれる複数の重鎖中の前記位置に存在する複数の特定のアミノ酸残基において機能性物質を有する結果、抗体が複数の機能性物質を有する、機能性物質を位置選択的に有する可溶性タンパク質またはその塩。
〔３８〕下記式（Ｖ）：
Ｆ－（Ｌ１－Ｂ）’－Ｒ’－Ｔ （Ｖ）
〔式中、
Ｌ１は、（ｉ’）生体直交性官能基を含む１価の基、または（ｉｉ’）生体直交性官能基を含まない１価の基であり、
Ｂは、（ａ）生体直交性官能基を含む２価の基、または（ｂ）生体直交性官能基を含まない２価の基であり、
（Ｌ１－Ｂ）’で表される構造単位は、機能性物質と、（ｉ’）および（ａ）における生体直交性官能基のいずれか一方または双方との間の反応により生成する部分を含む２価の構造単位であり、
Ｆは、機能性物質であり、
Ｒ’は、抗体のリジン残基と、リジン残基の側鎖に対して特異的な反応性基との間の反応により生成する部分であり、
Ｔは、抗体であり、
抗体は、連続する１～５０個のアミノ酸残基からなる標的領域中において特定のアミノ酸残基を１個以上含み、かつ、前記標的領域以外の非標的領域中において前記特定のアミノ酸残基を５個以上含む。〕で表され、かつＦ－（Ｌ１－Ｂ）’－Ｒ’で表される構造単位が、前記可溶性タンパク質の標的領域中に含まれる１個以上の特定のアミノ酸残基に対して３０％以上の位置選択性で結合している、機能性物質を位置選択的に有する可溶性タンパク質またはその塩。
〔３９〕前記可溶性タンパク質またはその塩が、下記式（Ｖ１）：
Ｌ１－Ｂ’（－Ｆ）－Ｒ’－Ｔ （Ｖ１）
〔式中、
Ｌ１、Ｆ、Ｒ’およびＴは、前記式（Ｖ）のものと同じであり、
Ｂ’は、機能性物質と生体直交性官能基との間の反応により生成する部分を含む２価の基である。〕で表され、かつＬ１－Ｂ’（－Ｆ）－Ｒ’で表される構造単位が、前記可溶性タンパク質の標的領域中に含まれる１個以上の特定のアミノ酸残基に対して３０％以上の位置選択性で結合している、機能性物質を位置選択的に有する可溶性タンパク質またはその塩である、〔３８〕の可溶性タンパク質またはその塩。
〔４０〕前記可溶性タンパク質またはその塩が、下記式（Ｖ２）：
Ｆ－Ｌ１’－Ｂ－Ｒ’－Ｔ （Ｖ２）
〔式中、
Ｆ、Ｂ、Ｒ’およびＴは、前記式（Ｖ）のものと同じであり、
Ｌ１’は、機能性物質と、（ｉ’）生体直交性官能基を含む１価の基との間の反応により生成する部分を含む２価の基である。〕で表され、かつＦ－Ｌ１’－Ｂ－Ｒ’で表される構造単位が、前記可溶性タンパク質の標的領域中に含まれる１個以上の特定のアミノ酸残基に対して３０％以上の位置選択性で結合している、機能性物質を位置選択的に有する可溶性タンパク質またはその塩、あるいは
下記式（Ｖ３）：
Ｆａ－Ｌ１’－Ｂ’（－Ｆｂ）－Ｒ’－Ｔ （Ｖ３）
〔式中、
Ｒ’およびＴは、前記式（Ｖ）のものと同じであり、
Ｌ１’は、前記式（Ｖ２）のものと同じであり、
Ｂ’は、機能性物質と生体直交性官能基との間の反応により生成する部分を含む２価の基であり、
ＦａおよびＦｂは、それぞれ、同一または異なる機能性物質である。〕で表され、かつＦａ－Ｌ１’－Ｂ’（－Ｆｂ）－Ｒ’で表される構造単位が、前記可溶性タンパク質の標
的領域中に含まれる１個以上の特定のアミノ酸残基に対して３０％以上の位置選択性で結合している、機能性物質を位置選択的に有する可溶性タンパク質またはその塩である、〔３８〕の可溶性タンパク質またはその塩。 Seventhly, the present invention provides a soluble protein or a salt thereof that regioselectively has a functional substance, and a method for producing the same.
(Soluble protein or its salt having a functional substance regioselectively)
[1] A soluble protein or a salt thereof regioselectively having a functional substance,
The soluble protein contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and the specific amino acid residue in a non-target region other than the target region 5 or more, and the functional substance binds to one or more specific amino acid residues contained in the target region with a position selectivity of 30% or more via a linker that does not contain a peptide portion. A soluble protein or a salt thereof that regioselectively has a functional substance.
[2] Formula (V) below:
F-(L1-B)'-R'-T (V)
[In the formula,
L1 is (i′) a monovalent group containing a bioorthogonal functional group or (ii′) a monovalent group not containing a bioorthogonal functional group;
B is (a) a divalent group that includes a bioorthogonal functional group or (b) a divalent group that does not include a bioorthogonal functional group;
The structural unit represented by (L1-B)' includes a moiety generated by a reaction between a functional substance and either one or both of the bioorthogonal functional groups in (i') and (a). is a divalent structural unit,
F is a functional substance,
R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein,
The soluble protein contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and the specific amino acid residue in a non-target region other than the target region 5 or more. ] and the structural unit represented by F-(L1-B)'-R' accounts for 30% or more of one or more specific amino acid residues contained in the target region of the soluble protein A soluble protein or a salt thereof regioselectively having a functional substance bound thereto with regioselectivity.
[3] the soluble protein or a salt thereof represented by the following formula (V1):
L1-B'(-F)-R'-T (V1)
[In the formula,
L1, F, R' and T are the same as in formula (V) above;
B' is a divalent group that includes a moiety generated by reaction between a functional agent and a bioorthogonal functional group. ] and the structural unit represented by L1-B'(-F)-R' accounts for 30% or more of one or more specific amino acid residues contained in the target region of the soluble protein The soluble protein or its salt of [2], which is a soluble protein or its salt that regioselectively has a functional substance bound with the regioselectivity of
[4] the soluble protein or a salt thereof represented by the following formula (V2):
FL1'-B-R'-T (V2)
[In the formula,
F, B, R' and T are the same as in formula (V) above;
L1' is a divalent group that includes a moiety produced by a reaction between a functional agent and (i') a monovalent group that includes a bioorthogonal functional group. ] and the structural unit represented by FL1′-BR′ is located at 30% or more of one or more specific amino acid residues contained in the target region of the soluble protein. A soluble protein or a salt thereof regioselectively bound to a functional substance, or the following formula (V3):
Fa-L1'-B'(-Fb)-R'-T (V3)
[In the formula,
R' and T are the same as in formula (V) above;
L1' is the same as in formula (V2) above,
B' is a divalent group that includes a moiety generated by a reaction between a functional agent and a bioorthogonal functional group;
Fa and Fb are the same or different functional substances, respectively. ] and a structural unit represented by Fa-L1′-B′(-Fb)-R′ for one or more specific amino acid residues contained in the target region of the soluble protein The soluble protein or salt thereof of [2], which is a soluble protein or a salt thereof regioselectively having a functional substance bound with a regioselectivity of 30% or more.
The soluble protein of any one of [1] to [4] or a salt thereof, wherein the soluble protein of [5] is a monoclonal antibody.
[6] The soluble protein of any one of [1] to [5], wherein the soluble protein is an IgG antibody, or a salt thereof.
[7] The soluble protein or salt thereof according to any one of [1] to [6], wherein the soluble protein is human-derived.
[8] any one of [1] to [7], wherein the soluble protein comprises any one Fc region protein selected from the group consisting of the following (A) to (C) and is an antibody having antigen-binding ability: Soluble protein or salt thereof:
(A) an Fc region protein comprising the amino acid sequence of SEQ ID NO: 1;
(B) an Fc region protein comprising an amino acid sequence in which one or several amino acid residues are inserted, added, deleted or substituted in the amino acid sequence of SEQ ID NO: 1; or (C) the amino acid sequence of SEQ ID NO: 1 and 90 An Fc region protein comprising an amino acid sequence exhibiting greater than or equal to % identity.
[9] The soluble protein or salt thereof of [1] to [8], wherein the target region is a region consisting of 1 to 10 consecutive amino acid residues.
[10] The soluble protein or salt thereof of [9], wherein the target region is a region consisting of 1 to 3 consecutive amino acid residues.
[11] The target region is (a) a region consisting of amino acid residues at positions 246-248 in the human IgG Fc region, (b) a region consisting of amino acid residues at positions 288-290 in the human IgG Fc region, or ( c) The soluble protein of [10] or a salt thereof, which is a region consisting of amino acid residues at position 317 in the human IgG Fc region.
[12] The soluble protein or salt thereof of [1] to [11], wherein the regioselectivity is 50% or more.
[13] The soluble protein or salt thereof of [12], wherein the regioselectivity is 70% or more.
[14] The soluble protein or salt thereof of [13], wherein the regioselectivity is 90% or more.
[15] the specific amino acid residue is a (where a is an arbitrary integer of 1 to 10 ) does not contain amino acid residues of the same type as the specific amino acid residue other than the specific amino acid residue present at the specific position in the region up to the remote position of the amino acid residue of [1] to [ 14] soluble protein or a salt thereof.
[16] the soluble protein is a multimeric protein comprising a plurality of monomeric proteins,
The multimeric protein has a plurality of functional substances as a result of having a functional substance at a plurality of specific amino acid residues present at the positions in a plurality of monomer proteins contained in the multimeric protein [1] , the soluble protein or a salt thereof according to any one of [5] to [15].
[17] the soluble protein is an antibody comprising a plurality of heavy chains;
[1], [5] to [16], wherein the antibody has a plurality of functional substances as a result of having a functional substance at a plurality of specific amino acid residues present at the positions in the plurality of heavy chains contained in the antibody. ] or a salt thereof.
[18] the soluble protein is a multimeric protein comprising a plurality of monomeric proteins,
T has a structural unit represented by ALBR' in a plurality of corresponding target regions in a plurality of monomeric proteins, so that the multimeric protein is ALB- A soluble protein or a salt thereof according to any one of [2] to [15], having a plurality of structural units represented by R'.
[19] the soluble protein is an antibody comprising a plurality of heavy chains;
T has a structural unit represented by ALBR' in a plurality of corresponding target regions in a plurality of heavy chains, such that the antibody is represented by ALBR'. a soluble protein or a salt thereof according to any one of [2] to [15] and [18], which has a plurality of structural units of
[20] The soluble protein of [17] or [19], which has two heavy chains, or a salt thereof.
[21] L1 is represented by the following formulas (L1-1) to (L1-2):
C1-Lb (L1-1)
C1 (L1-2)
[In the formula,
Lb is a divalent group,
C1 is a bioorthogonal functional group or a group other than a bioorthogonal functional group. ], the soluble protein of any one of [2] to [15] and [18] to [20] or a salt thereof.
[22] Lb is represented by the following formula (Lb'):

[In the formula,
p is any integer from 0 to 10,
q is any integer from 0 to 10,
X is a carbon atom, a nitrogen atom, or a single bond (wherein when X is a nitrogen atom, R _1b is absent; when X is a single bond, R _1a and R _1b are absent);
R _1a and R _1b are the same or different and are selected from the group consisting of a hydrogen atom or the substituents described above.
O (white circle) indicates binding to C1 and ● (filled circle) indicates binding to B. ], the soluble protein of [21] or a salt thereof.
[23] the bivalent group containing a bioorthogonal functional group is an azide residue, an aldehyde residue, a thiol residue, an alkyne residue, an alkene residue, a tetrazine residue, a nitrone residue, a hydroxylamine residue, a nitrile residue, hydrazine residue, ketone residue, boronic acid residue, cyanobenzothiazole residue, allyl residue, phosphine residue, maleimide residue, disulfide residue, thioester group, α-halocarbonyl residue, isonitrile residue The soluble protein or a salt thereof according to any one of [1] to [22], which is a divalent group containing a bioorthogonal functional group selected from the group consisting of groups, sydnon residues, and selenium residues in its main chain.
[24] the bivalent group containing a bioorthogonal functional group is an azide residue, an aldehyde residue, a thiol residue, an alkyne residue, an alkene residue, a halogen residue, a tetrazine residue, a nitrone residue, hydroxylamine; residue, nitrile residue, hydrazine residue, ketone residue, boronic acid residue, cyanobenzothiazole residue, allyl residue, phosphine residue, maleimide residue, disulfide residue, α-halocarbonyl residue, isonitrile The soluble protein or salt thereof according to any one of [1] to [22], which is a divalent group containing a bioorthogonal functional group selected from the group consisting of a residue, a sydnon residue and a selenium residue in its side chain.
[25] The bioorthogonal functional group is:

[In the formula,
R _1f , single or multiple R _1g and single or multiple R _1h are the same or different and are atoms or groups selected from the group consisting of (i) to (vii), or electron-withdrawing groups;
・ is a bond. ), the soluble protein or a salt thereof according to any one of [1] to [24].
[26] The divalent group (b) is optionally substituted alkylene, optionally substituted cycloalkylene, optionally substituted aryl, or optionally substituted divalent heterocyclic ring soluble of any of [1] to [25] selected from the group consisting of a group, -NR _a - (R _a represents a hydrogen atom or a substituent), -O-, or a combination of two or more thereof protein or its salt.
[27] B is the following formula (B-1):

[In the formula,
Y is -NH-, -O-, -CH ₂ -, or the following formula (B-2):

(In the formula,
V and V′ are the same or different and are —NH—, —O—, —CH ₂ —, or a single bond;
V is a divalent group containing a bioorthogonal functional group;
s is any integer from 0 to 10,
○ and ● in formula (B-2) have the same orientations as ○ and ● in formula (B-1), respectively. ) and
Z is an oxygen atom, a sulfur atom, or a hydrogen atom (when Z is a hydrogen atom, -C(=Z)- represents -CH ₂ -);
○ (white circle) in formula (B-1) indicates a bond to the L-side portion, and ● (black circle) indicates a bond to the R-side portion. ], a soluble protein or a salt thereof according to any one of [2] to [15] and [18] to [26].
[28] the reactive group is a reactive group specific to any one side chain of a lysine residue, a tyrosine residue, or a tryptophan residue, [2] to [15], [18] The soluble protein or salt thereof according to any one of ~[27].
[29] The soluble protein of any one of [2] to [15] and [18] to [28], wherein the reactive group is a reactive group specific to the side chain of a lysine residue, or a salt thereof .
[30] the reactive group is:

〔here,
R _5a and R _5c are atoms or groups selected from the group consisting of (i) to (vii);
R _5b is an electron withdrawing group,
j is any integer from 1 to 5;
k is any integer from 1 to 4. A soluble protein or a salt thereof according to any one of [2] to [15] and [18] to [29] corresponding to any one chemical structure selected from the group consisting of ].
[31] A bioorthogonal functional group is bound to a soluble protein via a linker having a main chain of 2 to 10 atoms that connects the functional substance and the side chain of a specific amino acid residue, [1 ] to [30] any soluble protein or a salt thereof.
[32] The bioorthogonal functional group is bound to the soluble protein via a linker in which the main chain connecting the functional substance and the side chain of a specific amino acid residue does not contain a ring structure, [1] to [31] ] or a salt thereof.
[33] The soluble protein or salt thereof according to any one of [2] to [15] and [18] to [32], wherein the main chain connecting the L1 terminal portion and R' has 2 to 10 atoms.
[34] The soluble protein or salt thereof according to any one of [2] to [15] and [18] to [33], wherein the main chain connecting A and R does not contain a ring structure.
[35] the partial structure represented by L1-B does not contain a peptide moiety, [2] to [15], [
18] to [34] any one of the soluble protein or a salt thereof.
[36] The soluble proteins or salts thereof regioselectively having a functional substance represented by the formulas (V1), (V2) and (V3) are the following (V1′), (V2′) and (V3'):

[In the formula,
C1 is a bioorthogonal functional group or a group other than a bioorthogonal functional group.
p, q, X, R _1a and R _1b are the same as in formula (Lb′) above;
Y' is a residue obtained by removing one hydrogen atom from Y in the formula (B-1),
Z is the same as in formula (B-1) above,
F, R' and T are the same as in formula (V) above. ],

[In the formula,
C1' is the moiety generated by the reaction between the functional substance and the bioorthogonal functional group.
p, q, X, R _1a and R _1b are the same as in formula (Lb′) above;
Y and Z are the same as in formula (B-1) above,
F, R' and T are the same as in formula (V) above. 〕,or

[In the formula,
C1' is the moiety generated by the reaction between the functional substance and the bioorthogonal functional group.
p, q, X, R _1a and R _1b are the same as in formula (Lb′) above;
Y' is a residue obtained by removing one hydrogen atom from Y in the formula (B-1),
Z is the same as in formula (B-1) above,
Fa and Fb are the same or different functional substances,
R' and T are the same as in formula (V) above. A soluble protein or a salt thereof according to any one of [2] to [15] and [18] to [35], which is a soluble protein or a salt thereof that regioselectively has a functional substance represented by ].
[37] A soluble protein or a salt thereof regioselectively having a functional substance,
The soluble protein contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and the specific amino acid residue in a non-target region other than the target region 5 or more, and the functional substance binds to one or more specific amino acid residues contained in the target region with a position selectivity of 30% or more via a linker that does not contain a peptide portion. and
the functional substance is attached to the soluble protein via the side chain of the lysine residue,
the soluble protein is an antibody comprising multiple heavy chains;
A soluble antibody regioselectively having a functional substance as a result of having a functional substance at a plurality of specific amino acid residues present at the above positions in a plurality of heavy chains contained in the antibody. protein or its salt.
[38] Formula (V) below:
F-(L1-B)'-R'-T (V)
[In the formula,
L1 is (i′) a monovalent group containing a bioorthogonal functional group or (ii′) a monovalent group not containing a bioorthogonal functional group;
B is (a) a divalent group that includes a bioorthogonal functional group or (b) a divalent group that does not include a bioorthogonal functional group;
The structural unit represented by (L1-B)' includes a moiety generated by a reaction between a functional substance and either one or both of the bioorthogonal functional groups in (i') and (a). is a divalent structural unit,
F is a functional substance,
R' is a moiety generated by reaction between a lysine residue of an antibody and a reactive group specific for the side chain of a lysine residue;
T is an antibody;
The antibody contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and 5 specific amino acid residues in a non-target region other than the target region. including more than ] and the structural unit represented by F-(L1-B)'-R' accounts for 30% or more of one or more specific amino acid residues contained in the target region of the soluble protein A soluble protein or a salt thereof regioselectively having a functional substance bound thereto with regioselectivity.
[39] the soluble protein or a salt thereof represented by the following formula (V1):
L1-B'(-F)-R'-T (V1)
[In the formula,
L1, F, R' and T are the same as in formula (V) above;
B' is a divalent group that includes a moiety generated by reaction between a functional agent and a bioorthogonal functional group. ] and the structural unit represented by L1-B'(-F)-R' accounts for 30% or more of one or more specific amino acid residues contained in the target region of the soluble protein The soluble protein of [38] or a salt thereof, which is a soluble protein or a salt thereof regioselectively having a functional substance that is regioselectively bound to
[40] the soluble protein or a salt thereof represented by the following formula (V2):
FL1'-B-R'-T (V2)
[In the formula,
F, B, R' and T are the same as in formula (V) above;
L1' is a divalent group that includes a moiety produced by a reaction between a functional agent and (i') a monovalent group that includes a bioorthogonal functional group. ] and the structural unit represented by FL1′-BR′ is located at 30% or more of one or more specific amino acid residues contained in the target region of the soluble protein. A soluble protein or a salt thereof regioselectively bound to a functional substance, or the following formula (V3):
Fa-L1'-B'(-Fb)-R'-T (V3)
[In the formula,
R' and T are the same as in formula (V) above;
L1' is the same as in formula (V2) above,
B' is a divalent group comprising a moiety generated by a reaction between a functional agent and a bioorthogonal functional group;
Fa and Fb are the same or different functional substances, respectively. ] and a structural unit represented by Fa-L1′-B′(-Fb)-R′ for one or more specific amino acid residues contained in the target region of the soluble protein The soluble protein or its salt of [38], which is a soluble protein or its salt regioselectively having a functional substance bound with a regioselectivity of 30% or more.

(Method for Producing a Soluble Protein Having a Functional Substance Regioselectively or a Salt Thereof)
The present invention also provides a method for producing a soluble protein or a salt thereof regioselectively having a functional substance, which requires specification by formula (V) or subordinate formulas thereof, among the soluble proteins of [1] to [40] above. I will provide a.
[41] A method for producing a soluble protein or a salt thereof regioselectively having a functional substance,
Formula (III) below:
ALB'(-F)-R'-T (III)
[In the formula,
A is an affinity substance for soluble proteins;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B' is a divalent group that includes a moiety generated by a reaction between a functional agent and a bioorthogonal functional group;
F is a functional substance,
R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein,
The soluble protein contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and the specific amino acid residue in a non-target region other than the target region 5 or more. ] and the structural unit represented by ALB'(-F)-R' is 30 for one or more specific amino acid residues contained in the target region of the soluble protein. cleaving the cleavable portion of a complex or a salt thereof regioselectively having an affinity substance for a soluble protein, a cleavable portion, a functional substance and a soluble protein bound with a regioselectivity of ≥%, or Formula (IV) below:
L1-B-R'-T (IV)
[In the formula,
L1 is (i′) a monovalent group containing a bioorthogonal functional group or (ii′) a monovalent group not containing a bioorthogonal functional group;
B is (a) a divalent group that includes a bioorthogonal functional group or (b) a divalent group that does not include a bioorthogonal functional group;
R' is a moiety generated by reaction between a soluble protein and a reactive group;
T contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and 5 of the specific amino acid residues in a non-target region other than the target region It is a soluble protein containing more than ] and the structural unit represented by ALB'(-F)-R' is 30 for one or more specific amino acid residues contained in the target region of the soluble protein. A soluble protein or a salt thereof regioselectively having a bioorthogonal functional group bound with a regioselectivity of 10% or more is reacted with a functional substance,
Formula (V) below:
F-(L1-B)'-R'-T (V)
[In the formula,
L1 is (i′) a monovalent group containing a bioorthogonal functional group or (ii′) a monovalent group not containing a bioorthogonal functional group;
B is (a) a divalent group that includes a bioorthogonal functional group or (b) a divalent group that does not include a bioorthogonal functional group;
The structural unit represented by (L1-B)' includes a moiety generated by a reaction between a functional substance and either one or both of the bioorthogonal functional groups in (i') and (a). is a divalent structural unit,
F is a functional substance,
R' and T are the same as in formula (III) or (IV) above. ] and the structural unit represented by F-(L1-B)'-R' accounts for 30% or more of one or more specific amino acid residues contained in the target region of the soluble protein producing a soluble protein or a salt thereof regioselectively having a functional substance regioselectively attached to

Preferably, the method of [41] above may be as follows.
[42] cleaving the cleavable portion of a complex or a salt thereof regioselectively comprising the affinity substance for the soluble protein, the cleavable portion, the functional substance and the soluble protein,
The following formula (V1):
L1-B'(-F)-R'-T (V1)
[In the formula,
L1 is (i′) a monovalent group containing a bioorthogonal functional group or (ii′) a monovalent group not containing a bioorthogonal functional group;
B', F, R' and T are the same as in formula (III) above. ] and the structural unit represented by L1-B'(-F)-R' accounts for 30% or more of one or more specific amino acid residues contained in the target region of the soluble protein The method of [41], which comprises producing a soluble protein or a salt thereof regioselectively having a functional substance that regioselectively binds to
[43] reacting a soluble protein or a salt thereof regioselectively having a bioorthogonal functional group with one or two functional substances,
The following formula (V2):
FL1'-B-R'-T (V2)
[In the formula,
B, R' and T are the same as in formula (IV) above;
L1' is a divalent group comprising a moiety produced by a reaction between a functional agent and (i') a monovalent group comprising a bioorthogonal functional group;
F is a functional substance. ] and the structural unit represented by FL1′-BR′ is located at 30% or more of one or more specific amino acid residues contained in the target region of the soluble protein. A soluble protein having a functional substance regioselectively bound thereto, or the following formula (V3):
Fa-L1'-B'(-Fb)-R'-T (V3)
[In the formula,
R' and T are the same as in formula (IV) above;
L1' is the same as in formula (V2) above,
B' is a divalent group that includes a moiety generated by a reaction between a functional agent and a bioorthogonal functional group;
Fa and Fb are the same or different functional substances, respectively. ] and a structural unit represented by Fa-L1′-B′(-Fb)-R′ for one or more specific amino acid residues contained in the target region of the soluble protein The method of [41], which comprises producing a soluble protein or a salt thereof regioselectively having a functional substance bound with a regioselectivity of 30% or more.
[44] the method of any one of [41] to [43], wherein the soluble protein is an antibody, and the reactive group is a reactive group specific to the side chain of a lysine residue;
[45] A method for producing a soluble protein or a salt thereof regioselectively having a functional substance,
(A) the following formula (II):
A-LB-R'-T (II)
[In the formula,
A is an affinity substance for soluble proteins;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B is (a) a divalent group comprising a bioorthogonal functional group;
R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein,
The soluble protein contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and the specific amino acid residue in a non-target region other than the target region 5 or more. ] and the structural unit represented by ALBR' is 30% or more position selection for one or more specific amino acid residues contained in the target region of the soluble protein reacting a functional substance with an affinity substance for a soluble protein and a soluble protein or a salt thereof regioselectively having a cleavable moiety,
Formula (III) below:
ALB'(-F)-R'-T (III)
[In the formula,
A, L, R' and T are the same as in formula (II) above;
B' is a divalent group that includes a moiety generated by a reaction between a functional agent and a bioorthogonal functional group;
F is a functional substance. ] and the structural unit represented by ALB'(-F)-R' is 30 for one or more specific amino acid residues contained in the target region of the soluble protein. (B) generating a complex or a salt thereof regioselectively having an affinity substance for a soluble protein, a cleavable moiety, a functional substance and a soluble protein bound with regioselectivity of greater than or equal to %; cleaving the cleavable portion of a complex or a salt thereof regioselectively having an affinity substance for a soluble protein, a cleavable portion, a functional substance and a soluble protein,
The following formula (V1):
L1-B'(-F)-R'-T (V1)
[In the formula,
L1 is (i′) a monovalent group containing a bioorthogonal functional group or (ii′) a monovalent group not containing a bioorthogonal functional group;
B', F, R' and T are the same as in formula (III) above. ] and the structural unit represented by L1-B'(-F)-R' accounts for 30% or more of one or more specific amino acid residues contained in the target region of the soluble protein producing a soluble protein or a salt thereof regioselectively having a functional substance regioselectively attached to
[46] A method for producing a soluble protein or a salt thereof regioselectively having a functional substance,
(A) the following formula (I):
A-L-B-R (I)
[In the formula,
A is an affinity substance for soluble proteins;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B is (a) a divalent group comprising a bioorthogonal functional group;
R is a reactive group for said soluble protein. ], a compound or a salt thereof regioselectively having an affinity substance, a cleavable moiety and a reactive group for a soluble protein represented by containing one or more specific amino acid residues in a target region consisting of residues, and containing five or more of the specific amino acid residues in a non-target region other than the target region),
Formula (II) below:
A-LB-R'-T (II)
[In the formula,
A, L, and B are the same as in formula (I) above R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein. ] and the structural unit represented by ALBR' is 30% or more position selection for one or more specific amino acid residues contained in the target region of the soluble protein producing a soluble protein or a salt thereof regioselectively having an affinity substance for a soluble protein and a cleavable moiety,
(B) reacting a substance having an affinity for the soluble protein and a soluble protein or a salt thereof regioselectively having a cleavable moiety with a functional substance,
Formula (III) below:
ALB'(-F)-R'-T (III)
[In the formula,
A and L are the same as in formula (I) above;
R' and T are the same as in formula (II) above;
B' is a divalent group that includes a moiety generated by a reaction between a functional agent and a bioorthogonal functional group;
F is a functional substance. ] and the structural unit represented by ALB'(-F)-R' is 30 for one or more specific amino acid residues contained in the target region of the soluble protein. (C) generating a complex or a salt thereof regioselectively having an affinity substance for a soluble protein, a cleavable moiety, a functional substance and a soluble protein bound with regioselectivity of ≥%; cleaving the cleavable portion of a complex or a salt thereof regioselectively having an affinity substance for a soluble protein, a cleavable portion, a functional substance and a soluble protein,
The following formula (V1):
L1-B'(-F)-R'-T (V1)
[In the formula,
L1 is (i′) a monovalent group containing a bioorthogonal functional group or (ii′) a monovalent group not containing a bioorthogonal functional group;
B', F, R' and T are the same as in formula (III) above. ] and the structural unit represented by L1-B'(-F)-R' accounts for 30% or more of one or more specific amino acid residues contained in the target region of the soluble protein producing a soluble protein or a salt thereof regioselectively having a functional substance regioselectively attached to
[47] A method for producing a soluble protein or a salt thereof regioselectively having a functional substance,
(A) the following formula (II):
A-LB-R'-T (II)
[In the formula,
A is an affinity substance for soluble proteins;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B is (a) a divalent group that includes a bioorthogonal functional group or (a) a divalent group that does not include a bioorthogonal functional group;
R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein,
The soluble protein contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and the specific amino acid residue in a non-target region other than the target region 5 or more. ] and the structural unit represented by ALBR' is 30% or more position selection for one or more specific amino acid residues contained in the target region of the soluble protein cleaving the cleavable portion of a soluble protein or a salt thereof regioselectively having an affinity substance for a soluble protein and a cleavable portion regioselectively bound to the soluble protein,
Formula (IV) below:
L1-B-R'-T (IV)
[In the formula,
B, R' and T are the same as in formula (II) above;
L1 is (i') a monovalent group that contains a bioorthogonal functional group or (ii') a monovalent group that does not contain a bioorthogonal functional group. ] and the structural unit represented by L1-BR' has a regioselectivity of 30% or more with respect to one or more specific amino acid residues contained in the target region of the soluble protein producing a soluble protein or salt thereof regioselectively having a bioorthogonal functional group attached thereto, and (B) a soluble protein or a salt thereof regioselectively bearing said bioorthogonal functional group; by reacting with a species or two or more functional substances,
The following formula (V2)
FL1'-B-R'-T (V2)
[In the formula,
B, R' and T are the same as in formula (IV) above;
L1' is a divalent group comprising a moiety produced by a reaction between a functional agent and (i') a monovalent group comprising a bioorthogonal functional group;
F is a functional substance. ] and the structural unit represented by FL1′-BR′ is located at 30% or more of one or more specific amino acid residues contained in the target region of the soluble protein. A soluble protein or a salt thereof regioselectively bound with a functional substance, or the following formula (V3):
Fa-L1'-B'(-Fb)-R'-T (V3)
[In the formula,
R' and T are the same as in formula (IV) above;
L1' is the same as in formula (V2) above,
B' is a divalent group that includes a moiety generated by a reaction between a functional agent and a bioorthogonal functional group;
Fa and Fb are the same or different functional substances, respectively. ] and a structural unit represented by Fa-L1′-B′(-Fb)-R′ for one or more specific amino acid residues contained in the target region of the soluble protein A method comprising producing a soluble protein or a salt thereof regioselectively having a functional substance bound with a regioselectivity of 30% or more.
[48] A method for producing a soluble protein or a salt thereof regioselectively having a functional substance,
(A) the following formula (I):
A-L-B-R (I)
[In the formula,
A is an affinity substance for soluble proteins;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B is (a) a divalent group that includes a bioorthogonal functional group or (a) a divalent group that does not include a bioorthogonal functional group;
R is a reactive group for said soluble protein. ] A compound or a salt thereof represented by a soluble protein (here, the soluble protein contains one or more specific amino acid residues in the target region consisting of 1 to 50 consecutive amino acid residues, and containing 5 or more of the specific amino acid residues in a non-target region other than the target region),
Formula (II) below:
A-LB-R'-T (II)
[In the formula,
A, L, and B are the same as in formula (I) above R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein. ] and the structural unit represented by ALBR' is 30% or more position selection for one or more specific amino acid residues contained in the target region of the soluble protein producing a soluble protein or a salt thereof regioselectively having an affinity substance for a soluble protein and a cleavable moiety,
(B) cleaving the affinity substance for the soluble protein and the cleavable portion of the soluble protein or salt thereof regioselectively having the cleavable portion,
Formula (IV) below:
L1-B-R'-T (IV)
[In the formula,
B, R' and T are the same as in formula (II) above;
L1 is (i') a monovalent group that contains a bioorthogonal functional group or (ii') a monovalent group that does not contain a bioorthogonal functional group. ] and the structural unit represented by L1-BR' has a regioselectivity of 30% or more with respect to one or more specific amino acid residues contained in the target region of the soluble protein producing a soluble protein or salt thereof regioselectively having a bioorthogonal functional group attached thereto, and (C) a soluble protein or a salt thereof regioselectively bearing said bioorthogonal functional group; by reacting with a species or two or more functional substances,
The following formula (V2):
FL1'-B-R'-T (V2)
[In the formula,
B, R' and T are the same as in formula (IV) above;
L1' is a divalent group comprising a moiety produced by a reaction between a functional agent and (i') a monovalent group comprising a bioorthogonal functional group;
F is a functional substance. ] and the structural unit represented by FL1′-BR′ is located at 30% or more of one or more specific amino acid residues contained in the target region of the soluble protein. A soluble protein or a salt thereof regioselectively bound with a functional substance, or the following formula (V3):
Fa-L1'-B'(-Fb)-R'-T (V3)
[In the formula,
R' and T are the same as in formula (IV) above;
L1' is the same as in formula (V2) above,
B' is a divalent group that includes a moiety generated by a reaction between a functional agent and a bioorthogonal functional group;
Fa and Fb are the same or different functional substances, respectively. ] and a structural unit represented by Fa-L1′-B′(-Fb)-R′ for one or more specific amino acid residues contained in the target region of the soluble protein A method comprising producing a soluble protein or a salt thereof regioselectively having a functional substance bound with a regioselectivity of 30% or more.

(I) The compounds of the present invention or salts thereof having an affinity substance for soluble proteins, a cleavable moiety and a reactive group are useful, for example, for regioselective modification of soluble proteins.
(II) an affinity substance for a soluble protein and a soluble protein of the present invention or a salt thereof having (regioselectively) a cleavable moiety, (III) an affinity substance for a soluble protein, a cleavable moiety, a functional substance and a soluble The complex of the present invention having a protein (regioselectively) or a salt thereof, and (IV) the soluble protein of the present invention having a bioorthogonal functional group (regioselectively) or a salt thereof are functional substances, for example (regioselectively) as an intermediate for the preparation of soluble proteins or salts thereof.
(V) The soluble protein or salt thereof of the present invention having (regioselectively) a functional substance is useful, for example, as a drug or reagent (eg, diagnostic agent, research reagent). In particular, when the soluble protein is an antibody, the antibody or salt thereof of the present invention having a functional substance (regioselectively) is suitable for these uses.

FIG. 1-1 shows the position of soluble proteins (eg, antibodies) by the compounds of the present invention [compounds having an affinity substance for soluble proteins, a cleavable moiety and a reactive group: ALBR (I)]. FIG. 1 is a schematic diagram (Part 1) showing the concept of selective modification. First, the compounds of the present invention associate with soluble proteins (T), such as antibodies, via an affinity substance (A) for soluble proteins. Next, the compound of the present invention is applied via a reactive group (R) (activated ester in the figure) to a specific amino acid residue in the target region that exists near the site of association between the affinity substance and the soluble protein. A conjugate of the compound of the present invention with a soluble protein [a structural unit containing an affinity substance for a soluble protein and a cleavable moiety is position-selected soluble protein: ALBR'-T(II)]. FIG. 1-2 is a schematic diagram (No. 2) showing the concept of regioselective modification of soluble proteins (eg, antibodies) by the compounds of the present invention. Cleavage of the cleavable moiety in the linker (L) produces a soluble protein (eg, antibody) regiospecifically modified with bioorthogonal functional groups. 1-3 are schematic diagrams (No. 3) showing the concept of regioselective modification of soluble proteins (eg, antibodies) by the compounds of the present invention. Reaction of bioorthogonal functional groups with functional agents (eg, drugs) produces soluble proteins (eg, antibodies) regiospecifically modified with functional agents. FIG. 2 is a diagram showing the relationship of each invention of the present invention (notation of salt is omitted). In reaction (1), a compound having an affinity for a soluble protein, a cleavable moiety and a reactive group is reacted with the soluble protein to produce a soluble protein having an affinity for the soluble protein and a cleavable moiety. In reaction (2), a soluble protein having an affinity for the soluble protein and a cleavable moiety is reacted with a functional agent to provide a conjugate having an affinity for the soluble protein, the cleaving moiety, the functional agent and the soluble protein. generate a body. In reaction (3), the cleavable portion of the complex with the affinity agent for the soluble protein, the cleavable moiety, the functional agent and the soluble protein is cleaved to produce a soluble protein with the functional agent (wherein A portion containing the affinity substance is produced as a by-product). In reaction (4), the affinity agent for the soluble protein and the cleavable portion of the soluble protein with the cleavable moiety are cleaved to produce a soluble protein with a bioorthogonal functional group (wherein the affinity agent containing portion is produced as a by-product). In reaction (5), a soluble protein bearing a bioorthogonal functional group is reacted with a functional agent to produce a soluble protein bearing the functional agent. Reactions (2) and (5) can be carried out analogously. Reactions (3) and (5) can also be carried out analogously. FIG. 3 shows HIC-HPLC analysis (detection: 225 nm) of anti-HER2 IgG antibody trastuzumab specifically modified with a peptide reagent (peptide disulfide linker conjugate-NHS activator). The samples were reacted under the following conditions. a: Trastuzumab + peptide reagent 12 equivalents (solvent replacement with Amicon 10K after reaction); b: Trastuzumab + peptide reagent 12 equivalents; c: Trastuzumab + peptide reagent 6 equivalents; d: Trastuzumab raw material; e: peptide reagent only; : DMF only. FIG. 4 shows HIC-HPLC analysis (detection: 280 nm) of anti-HER2 IgG antibody trastuzumab specifically modified with a peptide reagent (peptide disulfide linker conjugate-NHS activator). The samples were reacted under the same conditions as in FIG. FIG. 5 shows HIC-HPLC analysis (detection: 225 nm) of the anti-CD20 antibody rituximab specifically modified with a peptide reagent (peptide disulfide linker conjugate-NHS activator). The samples were reacted under the following conditions. a: Rituximab + peptide reagent 12 equivalents (solvent replacement with Amicon 10K after reaction); b: Rituximab + peptide reagent 12 equivalents; c: Rituximab + peptide reagent 6 equivalents; d: Rituximab raw material; e: peptide reagent only; : DMF only. FIG. 6 shows HIC-HPLC analysis (detection: 280 nm) of the anti-CD20 antibody rituximab specifically modified with a peptide reagent (peptide disulfide linker conjugate-NHS activator). The samples were reacted under the following conditions. a: Rituximab + peptide reagent 12 equivalents (solvent replacement with Amicon 10K after reaction); b: Rituximab + peptide reagent 12 equivalents; c: Rituximab + peptide reagent 6 equivalents; d: Rituximab raw material; e: peptide reagent only; : DMF only. FIG. 7 shows analysis by RP-HPLC (detection: 225 nm and 280 nm) of products obtained by linker cleavage and reoxidation of trastuzumab peptide conjugates. a: trastuzumab; b: trastuzumab + peptide reagent 6 equivalents; c: trastuzumab reduced with DTT; d: buffer pH 8.0, reduced with D,L-dithiothreitol at an antibody concentration of 72 μM; e: d dehydroascorbic acid f: 24 hours after reoxidation of d with dehydroascorbic acid. FIG. 8 shows (1) the amino acid sequence of the heavy chain of trastuzumab whose sugar chain has been cleaved with PNGase, (2) the amino acid sequence of the IgG1 Fc region whose sugar chain has been cleaved with PNGase, and (3) the amino acid sequence of the light chain of trastuzumab. It is a figure which shows. Figure 9 shows the MS spectrum (m /z 998.14796, trivalent). FIG. 10 shows the CID spectrum of a peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPK (SEQ ID NO: 5) containing the site of modification to a lysine residue by tryptic digestion of trastuzumab (thiol derivative (+146.004 Da) undergoing carboxymethylation with iodoacetic acid). It is a diagram. FIG. 11 shows the MS spectrum of a peptide fragment of LLGGPSVFLFPPKPKD (SEQ ID NO: 6) containing a modification site to a lysine residue by Glu-C digestion of trastuzumab (thiol introduction (+146.004 Da) subjected to carboxymethylation by iodoacetic acid). (m/z 929.49651, divalent). FIG. 12 shows the CID spectrum of a peptide fragment of LLGGPSVFLFPPKPKD (SEQ ID NO: 6) containing a modification site to a lysine residue by Glu-C digestion of trastuzumab (thiol introduction (+146.004 Da) subjected to carboxymethylation by iodoacetic acid). It is a figure which shows. FIG. 13 shows a peptide fragment (m/ Fig. 10 shows the MS spectrum of Z 994.12546, trivalent). FIG. 14 shows the CID spectrum of a peptide fragment of THTCPPCPAPEAEEGAPSVFLFPPKPK (SEQ ID NO: 7) containing the site of modification to a lysine residue by tryptic digestion of IgG1 Fc (thiol introduction (+146.004 Da) subjected to carboxymethylation with iodoacetic acid). FIG. 4 is a diagram showing; FIG. 15 shows the MS of a peptide fragment of GAPSVFLFPPKPKKDTLMISRTPE (SEQ ID NO: 8) containing the site of modification of IgG1 Fc to a lysine residue by Glu-C digestion (Carboxymethylated with iodoacetic acid (+146.004 Da)). Fig. 3 shows the spectrum (m/z 892.12624, trivalent); FIG. 16 shows the CID of a peptide fragment of GAPSVFLFPPKPKKDTLMISRTPE (SEQ ID NO: 8) containing the site of modification to a lysine residue by Glu-C digestion of IgG1 Fc (thiol introduction (+146.004 Da) subjected to Carboxymethylation with iodoacetic acid). FIG. 4 is a diagram showing spectra; Figure 17 shows the Fc region in the heavy chain of trastuzumab and the consensus amino acid sequence (SEQ ID NO: 1) of the IgG1 Fc region. FIG. 18 shows the identified amino acid sequences and modifications for modified trastuzumab (IgG1). The gray area is the identified amino acid sequence. Also, the identified modifications are noted above the amino acid sequence. Regioselective azide-introduced modification was confirmed only at the two enclosed lysine residues (positions 246 and 248 according to EU numbering). FIG. 19 is a diagram showing the modification site of the azide derivative of trastuzumab. PSMs (Peptide Spectrum Matches) are the number of times a spectrum matching the corresponding peptide was observed, and the higher the number, the higher the probability. By applying a signal intensity filter, modification was identified only at the 246-position lysine residue as in (2). Modification sites identified only in (1) are likely to be noise. FIG. 20 shows the CID spectra of peptides containing the modification site of the azide introduction of trastuzumab. Corresponds to the CID spectrum of the peptide in the boxed portion in FIG. Spectra with m/z consistent with theoretical values are shown. FIG. 21 shows b/y ions with identified CID spectra. The b/y ions identified in FIG. 20 are shown. Since the b25 ion has been identified, it can be seen that there is a high possibility that position 246 is modified by EU numbering. FIG. 22 shows area value comparison of modified trastuzumab with unmodified trastuzumab. Based on the prediction that the area value of the unmodified peptide would be smaller at the site where modification occurred, when compared with the area value obtained with unmodified trastuzumab, peptides containing lysine residues at positions 246 and 248 by EU numbering The area value decreased significantly only in FIG. 23 shows analysis of the DAR of modified trastuzumab by Q-TOFMS. Average DAR was 2 from peak observations. FIG. 24 is a diagram showing amino acid sequence data used for searching. Since it is known that deglycosylation changes N at the 300th residue of the heavy chain of trastuzumab to D, two heavy chain amino acid sequences were used for analysis. FIG. 25 shows the MS spectrum of the peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 40) containing the site of modification (azido introduction (+254.102 Da)) to a lysine residue by tryptic digestion of trastuzumab (observed: m/z 979. 49982, theoretical value: 979.49975, tetravalent). FIG. 26 shows the CID spectrum of a peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 40) containing the modification site (azido introduction (+254.102 Da)) to a lysine residue by tryptic digestion of trastuzumab. FIG. 27 shows the results of HIC-UPLC analysis of specific modification of trastuzumab (detection wavelength: UV225 nm or UV280 nm) (Example 16). FIG. 28 shows the results of HIC-UPLC analysis of specific modification of trastuzumab (detection wavelength: UV225 nm or UV280 nm) (Example 17). FIG. 29 shows the results of HIC-UPLC analysis of specific modification of trastuzumab (detection wavelength: UV225 nm or UV280 nm) (Example 18). FIG. 30 shows the results of HIC-UPLC analysis of specific modification of trastuzumab (detection wavelength: UV225 nm or UV280 nm) (Example 19). FIG. 31 shows the results of HIC-UPLC analysis of specific modification of trastuzumab (detection wavelength: UV225 nm or UV280 nm) (Example 20). FIG. 32 shows the results of HIC-UPLC analysis of specific modification of trastuzumab (detection wavelength: UV225 nm or UV280 nm) (Example 21). FIG. 33 shows the results of HIC-UPLC analysis of specific modification of trastuzumab (detection wavelength: UV225 nm or UV280 nm) (Example 22). FIG. 34 shows the results of HIC-UPLC analysis of specific modification of trastuzumab (detection wavelength: UV225 nm or UV280 nm) (Example 23). FIG. 35 shows the results of HIC-UPLC analysis of specific modification of trastuzumab (detection wavelength: UV225 nm or UV280 nm) (Example 24). FIG. 36 shows the results of HIC-UPLC analysis of specific modification of trastuzumab (detection wavelength: UV225 nm or UV280 nm) (Example 25). FIG. 37 shows the results of HIC-UPLC analysis of specific modification of trastuzumab (detection wavelength: UV225 nm or UV280 nm) (Example 26). FIG. 38 shows the results of HIC-UPLC analysis of specific modification of trastuzumab (detection wavelength: UV225 nm or UV280 nm) (Example 27). FIG. 39 shows the results of HIC-UPLC analysis of specific modification of trastuzumab (detection wavelength: UV225 nm or UV280 nm) (Example 28). FIG. 40 shows the results of HIC-UPLC analysis of specific modification of trastuzumab (detection wavelength: UV225 nm or UV280 nm) (Example 29). FIG. 41 shows the results of HIC-UPLC analysis of specific modification of trastuzumab (detection wavelength: UV225 nm or UV280 nm) (Example 30). Figure 42 shows the results of HIC-UPLC analysis of specific modification of trastuzumab (detection wavelength: UV 280 nm) (Example 31). AU on the vertical axis indicates absorbance (the same applies to subsequent drawings). Figure 43 shows the results of HIC-UPLC analysis of specific modification of trastuzumab (detection wavelength: UV 280 nm) (Example 32). FIG. 44 shows the results of HIC-UPLC analysis (detection wavelength: UV280 nm) of specific modification of trastuzumab (Example 33). Figure 45 shows the results of HIC-UPLC analysis of specific modification of trastuzumab (detection wavelength: UV 280 nm) (Example 34). Figure 46 shows the results of HIC-UPLC analysis of specific modification of trastuzumab (detection wavelength: UV 280 nm) (Example 35). Figure 47 shows the results of HIC-UPLC analysis of specific modification of trastuzumab (detection wavelength: UV 280 nm) (Example 36). Figure 48 shows the results of HIC-UPLC analysis of specific modification of trastuzumab (detection wavelength: UV 280 nm) (Example 37). Figure 49 shows the results of HIC-UPLC analysis (detection wavelength: UV 280 nm) of specific modification of trastuzumab (Example 38). FIG. 50 shows the results of HIC-UPLC analysis (detection wavelength: UV280 nm) of specific modification of trastuzumab (Example 39). FIG. 51 shows VVSVLTVLHQDWLNGKEYK (VVSVLTVLHQDWLNGKEYK ( 66) shows the MS spectrum (m/z 791.74872, trivalent) of the peptide fragment of SEQ ID NO: 66). FIG. 52 shows VVSVLTVLHQDWLNGKEYK (VVSVLTVLHQDWLNGKEYK ( 66) shows the CID spectrum of the peptide fragment of SEQ ID NO: 66). FIG. 53 shows EEQYDSTYRVVSVLTVLHQDWLNGKEYK (EQYDSTYRVVSVLTVLHQDWLNGKEYK) containing the site of modification to a lysine residue by tryptic digestion of trastuzumab modified with the binder peptide of Example 39 (thiol derivative (+145.019 Da) after carbamidomethylation with iodoacetamide). 67) MS spectrum (m/z 886.93408, tetravalent) of the peptide fragment of SEQ ID NO: 67). FIG. 54 shows EEQYDSTYRVVSVLTVLHQDWLNGKEYK (EQDWLNGKEYK) containing the site of modification to a lysine residue by trypsin digestion of trastuzumab modified with the binder peptide of Example 39 (thiol derivative (+145.019 Da) after Carbamidomethylation with iodoacetamide). 67) shows the CID spectrum of the peptide fragment of SEQ ID NO: 67). Figure 55 shows the results of HIC-UPLC analysis of specific modification of trastuzumab (detection wavelength: UV 280 nm) (Example 40). FIG. 56 depicts FNWYVDGVEVHNAKTKPR (thiol derivatization (+145.019 Da) with iodoacetamide Carbamidomethylation) containing the site of modification to a lysine residue by tryptic digestion of trastuzumab modified with the binder peptide of Example 40 ( 69) MS spectrum (m/z 769.04688, trivalent) of the peptide fragment of SEQ ID NO: 69). FIG. 57 depicts FNWYVDGVEVHNAKTKPR (thiol derivatization (+145.019 Da) with iodoacetamide carbamidomethylation) containing the site of modification to a lysine residue by trypsin digestion of trastuzumab modified with the binder peptide of Example 40 ( 69) shows the CID spectrum of the peptide fragment of SEQ ID NO: 69). Figure 58 shows the results of HIC-UPLC analysis of specific modification of trastuzumab (detection wavelength: UV 280 nm) (Example 41). FIG. 59 shows THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 40) containing the site of tryptic digestion of trastuzumab modified with a thiol derivatization to a lysine residue (thiol derivatization (+145.019 Da) Carbamidomethylated with iodoacetamide). ) shows the MS spectrum (m/z 952.23145, tetravalent) of the peptide fragment of ). FIG. 60 shows THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 40) containing the site of modification to a lysine residue by trypsin digestion of trastuzumab modified with a thiol derivatization (thiol derivatization (+145.019 Da) Carbamidomethylated with iodoacetamide). ) shows the CID spectrum of the peptide fragment of . Figure 61 shows the results of HIC-UPLC analysis of specific modification of trastuzumab (detection wavelength: UV 280 nm) (Example 42). FIG. 62 shows THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 40) containing the site of modification to a lysine residue by trypsin digestion of trastuzumab modified with a thiol derivatization (thiol derivatization (+145.019 Da) Carbamidomethylated with iodoacetamide). ) shows the MS spectrum (m/z 952.22968, tetravalent) of the peptide fragment of ). FIG. 63 shows THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 40) containing the site of tryptic digestion of trastuzumab modified with a thiol derivatization to a lysine residue (thiol derivatization (+145.019 Da) Carbamidomethylated with iodoacetamide). ) shows the CID spectrum of the peptide fragment of . Figure 64 shows the results of HIC-UPLC analysis of specific modification of trastuzumab (detection wavelength: UV 280 nm) (Example 43). FIG. 65 depicts FNWYVDGVEVHNAKTKPR (SEQ ID NO: 69) containing the site of modification to a lysine residue by tryptic digestion of trastuzumab modified with a thiol derivatization (thiol derivatization (+145.019 Da) Carbamidomethylated with iodoacetamide). ) (m/z 769.04529, trivalent) of the peptide fragment. FIG. 66 depicts FNWYVDGVEVHNAKTKPR (SEQ ID NO: 69) containing the site of modification to a lysine residue by trypsin digestion of trastuzumab modified with a thiol derivatization (thiol derivatization (+145.019 Da) Carbamidomethylated with iodoacetamide). ) shows the CID spectrum of the peptide fragment of . FIG. 67 shows the results of HIC-UPLC analysis (detection wavelength: UV 280 nm) of specific modification of human IgG2 antibody (Example 2-2). FIG. 68 shows the results of HIC-UPLC analysis (detection wavelength: UV 280 nm) of specific modification of human IgG2 antibody (Example 43). FIG. 69 shows the results of HIC-UPLC analysis (detection wavelength: UV 280 nm) of specific modification of human IgG4 antibody (Example 2-2). FIG. 70 shows the results of HIC-UPLC analysis (detection wavelength: UV 280 nm) of specific modification of human IgG4 antibody (Example 43).

1. A compound or salt thereof containing an affinity substance for a soluble protein, a cleavable moiety and a reactive group 1-1. SUMMARY The present invention provides compounds or salts thereof of formula (I) that include an affinity agent for soluble proteins, a cleavable moiety and a reactive group.
A-L-B-R (I)
[In the formula,
A is an affinity substance for soluble proteins;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B is (a) a divalent group that includes a bioorthogonal functional group or (b) a divalent group that does not include a bioorthogonal functional group;
R is a reactive group for said soluble protein. ]

In formula (I) and other formulas presented in connection with the present invention, a - (hyphen) indicates that the two units on either side of it are covalently linked. Thus, in formula (I), A is covalently bonded to L, L is covalently bonded to A and B, B is covalently bonded to L and R, and R is covalently bonded to B. are covalently bonded.

1-2. Affinity substance for soluble protein (A)
In formula (I), A is an affinity substance for soluble proteins. An affinity substance is a substance that has the ability to bind to a target by non-covalent bonding.

The affinity agents used in the present invention target soluble proteins, also called secreted proteins. Examples of such soluble proteins include antibodies, soluble receptors, ligands, albumin, erythropoietin (EPO), vascular endothelial growth factor (Anti-VEGF), bone morphogenetic proteins, follicle stimulating hormone (FSH), glucagon, granules, colony-stimulating factor, granular macrophage colony-stimulating factor, ciliary gonadotrobin, insulin, interleukin, interferon, platelet-derived growth factor (PDGF), growth factor (TGF family, FGF family). Soluble proteins may be proteins modified with biomolecules (eg sugars) (eg glycoproteins) or unmodified with biomolecules.

Soluble proteins that are targets of affinity agents are also naturally occurring or artificial proteins.

Naturally derived proteins include, for example, proteins derived from organisms and viruses. Examples of biological proteins include proteins derived from mammals, animals such as birds (eg, chickens), insects, microorganisms, plants, fungi, and fish. Preferably, the naturally occurring protein is a mammalian protein. Examples of mammals include primates (e.g., humans, monkeys, chimpanzees), rodents (e.g., mice, rats, guinea pigs, hamsters, rabbits), pets (e.g., dogs, cats), livestock (e.g., cattle, pigs, goats), working animals (eg horses, sheep). The naturally occurring protein is more preferably a primate- or rodent-derived protein, and even more preferably a human-derived protein from the viewpoint of clinical application of the present invention. Virus-derived proteins include, for example, influenza virus (eg, avian influenza virus, swine influenza virus), AIDS virus, Ebola virus, and phage virus.

Artificial proteins include, for example, modified proteins of naturally occurring proteins (e.g., proteins in which one or more amino acid residue mutations selected from the group consisting of substitutions, deletions and insertions are introduced into naturally occurring proteins), fusion proteins, and artificially designed monoclonal antibodies. Artificially designed monoclonal antibodies include, for example, chimeric antibodies, humanized antibodies, human antibodies, antibodies to which a predetermined sugar chain has been added (e.g., a sugar chain-binding consensus sequence such as an N-type sugar chain-binding consensus sequence). antibodies engineered to have), bispecific antibodies, scFv antibodies, Fab antibodies, F(ab') ₂ antibodies, VHH antibodies, Fc region proteins, Fc fusion proteins.

The soluble protein that is the target of the affinity agent may also be a monomeric protein or a multimeric (eg, dimer, trimer, or tetramer) protein. When the protein that is the target of the affinity substance is a multimeric protein, the multimeric protein is a homomultimer or a heteromultimer. A multimeric protein is a protein that forms a multimer via a covalent bond (e.g., disulfide bond) (e.g., an antibody having a structure in which two units consisting of a light chain and a heavy chain are linked via a disulfide bond. ) or proteins that form multimers through non-covalent bonds (ie association), but proteins that form multimers through covalent bonds are preferred. Multimeric proteins that are targets of affinity substances include, for example, bivalent antibodies (eg, IgG, IgD, IgE), tetravalent or higher antibodies (eg, IgA antibodies, IgM antibodies), and albumin.

The soluble protein that is the target of the affinity substance may also be composed of any amino acid residue, but preferably is composed of the 20 naturally occurring L-α-amino acid residues that normally constitute proteins. . Such amino acid residues include, for example, L-alanine (A), L-asparagine (N), L-cysteine (C), L-glutamine (Q), L-isoleucine (I), L-leucine ( L), L-methionine (M), L-phenylalanine (F), L-proline (P), L-serine (S), L-threonine (T), L-tryptophan (W), L-tyrosine (Y ), L-valine (V), L-aspartic acid (D), L-glutamic acid (E), L-arginine (R), L-histidine (H), or L-lysine (K), and glycine (G ) (hereinafter, the notation of L is omitted). A soluble protein may be composed of, for example, 100 or more, preferably 120 or more, more preferably 150 or more, still more preferably 180 or more, particularly preferably 200 or more amino acid residues. Soluble proteins may also be, for example, 1000 or less, preferably 900 or less, more preferably 800 or less, even more preferably 700 or less, particularly preferably 600 or less. More specifically, soluble proteins are, for example, 100 to 1000, preferably 120 to 900, more preferably 150 to 800, even more preferably 180 to 700 or more, particularly preferably 200 to 600 amino acids. It may be composed of residues. When the soluble protein is an antibody (eg, an artificially designed monoclonal antibody as described above), the above number of amino acid residues may correspond to the amino acid residues of the heavy chain of the antibody.

The soluble protein that is the target of the affinity substance further includes a specific amino acid residue having a side chain or terminus (N-terminus and/or C-terminus), preferably a side chain, with which a reactive group as described below can react. It is a protein comprising at a position or at multiple positions (preferably at multiple positions). Such specific amino acid residues include, for example, the 14 amino acid residues described below, preferably the group consisting of lysine, tyrosine, tryptophan, and cysteine residues. It is an amino acid residue selected from In view of the ability of the compounds of the present invention to regioselectively modify soluble proteins, soluble proteins containing such specific amino acid residues at multiple positions are preferred. The plurality of positions is not particularly limited as long as it is 2 or more positions, but for example, 3 or more positions, preferably 5 or more positions, more preferably 10 or more positions, still more preferably 20 or more positions, particularly preferably may be 30 or more positions. The plurality of positions may also be, for example, 200 positions or less, preferably 180 positions or less, more preferably 150 positions or less, even more preferably 120 positions or less, particularly preferably 100 positions or less. More specifically, the plurality of positions is, for example, 3 to 200 positions, preferably 5 to 180 positions, more preferably 10 to 150 positions, even more preferably 20 to 120 positions, particularly preferably 30 to There may be 100 positions. Even in a soluble protein containing such specific amino acid residues at multiple positions, the compounds of the present invention can regioselectively modify a specific amino acid residue present at a specific position. . For example, the number of lysine residues in human IgG1 is generally said to be about 70-90, depending on the amino acid composition in the variable region. The present invention has succeeded in regioselectively modifying a lysine residue present at such a specific position in human IgG1.

More specifically, in the present invention, amino acid residues present at specific positions in proteins while maintaining the functions of proteins such as antibodies (that is, maintaining native folding without denaturing the proteins) From the viewpoint of modification, site-selective modification of amino acid residues exposed on the protein surface is preferred. For example, in human IgG, such as human IgG1, exposed lysine residues and exposed tyrosine residues are present at the following positions (according to EU numbering; see http://www.imgt.org/IMGTScientificChart/Numbering/Hu_IGHGnber.html reference). (1) exposed lysine residue CH2 domain (positions 246, 248, 274, 288, 290, 317, 320, 322, 338)
CH3 domain (positions 360, 414, 439)
(2) exposed tyrosine residue CH2 domain (278, 296, 300)
CH3 domain (position 436)
Therefore, when human IgG, such as human IgG1, is modified at lysine or tyrosine residues, modification at these positions is preferred.

Preferably, when human IgG such as human IgG1 is modified with a lysine residue or a tyrosine residue, among the positions (1) and (2) above, the following positions with high surface exposure are present: Lysine or tyrosine residues may be modified.
(1′) exposed lysine residues CH2 domain (positions 246, 248, 274, 288, 290, 317, 320, 322)
CH3 domain (positions 360, 414, 439)
(2′) exposed tyrosine residues CH2 domain (positions 278, 296, 300)
CH3 domain (position 436)
Therefore, when human IgG, such as human IgG1, is modified at lysine or tyrosine residues, modification at these positions is more preferred.

More preferably, when human IgG such as human IgG1 is modified with a lysine residue, among the positions of (1) above, a predetermined position in the CH2 domain that can be efficiently modified in the present invention (e.g. , 246, 248, 288, 290, 317) may be modified.

In certain embodiments, when the soluble protein targeted by the affinity substance contains specific amino acid residues at multiple positions as described above, in the target region consisting of 1 to 50 consecutive amino acid residues, It may contain one or more specific amino acid residues and five or more specific amino acid residues in a non-target region other than the target region. The target regions are preferably 1-30, more preferably 1-20, even more preferably 1-10, 1-5 or 1-3 (ie 1, 2 or 3 ) may consist of amino acid residues. Particularly preferably, the target region may be a region consisting of specific amino acid residues at specific positions. Such a specific position varies depending on the type of target protein and affinity substance, and is, for example, a specific position in a specific region (e.g., CH1, CH2, CH3) in the constant region of an antibody. may also be located, preferably in CH2 of the antibody. More specifically, the target region is Eu
It may be the following residues according to numbering:
(1) Lys248 residue (hereinafter simply referred to as “Lys248” in this specification and corresponds to the 18th residue of the human IgG CH2 region (SEQ ID NO: 1)) or Lys246 residue (hereinafter referred to as “Lys248” in this specification) Also simply referred to as "Lys246", it corresponds to the 16th residue of the human IgG CH2 region (SEQ ID NO: 1))
:
(2) Lys288 residue (hereinafter simply referred to as “Lys288” in this specification, corresponding to the 58th residue of the human IgG CH2 region (SEQ ID NO: 1)) or Lys290 residue (hereinafter referred to as “Lys288” in this specification) also simply referred to as "Lys290", corresponding to the 60th residue of the human IgG CH2 region (SEQ ID NO: 1);
(3) Lys317 residue (hereinafter also simply referred to as “Lys317” in this specification, which corresponds to the 87th residue of the human IgG CH2 region (SEQ ID NO: 1)).

According to the present invention, specific amino acid residues in the target region can be highly regioselectively modified. Such regioselectivity is, for example, 30% or more, preferably 40% or more, more preferably 50% or more, even more preferably 60% or more, particularly preferably 70%.
80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% or more.

The target region also has a specific amino acid residue present at a specific position on the N-terminal side and the C-terminal side of the specific position (where a is any number from 1 to 10). (which is an integer of ) does not contain amino acid residues of the same type as the specific amino acid residue other than the specific amino acid residue present at the specific position in the region to the remote position of the amino acid residue good too. a is preferably an integer of 1 to 5, more preferably an integer of 1 to 3, even more preferably 1 or 2, particularly preferably 1;

In a preferred embodiment, the affinity agent for soluble proteins is an affinity agent for antibodies. Antibodies are polyclonal antibodies or monoclonal antibodies. Antibody isotypes include, for example, IgG (eg, IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, IgE, and IgY. Antibodies are full length antibodies or antibody fragments (eg, F(ab') ₂ , Fab', Fab, Fv, single chain antibodies), but full length antibodies are preferred.

An antibody is an antibody against any antigen. For example, such antigens may be components found in organisms or viruses as described above. Such antigens also include, for example, proteins [oligopeptides, polypeptides. proteins modified with biomolecules such as sugars (eg, glycoproteins)], sugar chains, nucleic acids, and low-molecular-weight compounds.

Preferably, the antibody may be an antibody whose antigen is a protein. Proteins include, for example, cell membrane receptors, cell membrane proteins other than cell membrane receptors (eg, extracellular matrix proteins), ligands, and soluble receptors.

More specifically, the protein that is the antigen of the antibody may be a disease target protein. Disease target proteins include, for example:

(1) Cancer area PD-L1, GD2, PDGFRα (platelet-derived growth factor receptor), CD22, HER2, phosphatidylserine (PS), EpCAM, fibronectin, PD-1, VEGFR-2, CD33, HGF, gpNMB, CD27, DEC-205, folate receptor, CD37, CD19, Trop2, CEACAM5, S1P, HER3, IGF-1R, DLL4, TNT-1/B, CPAAs, PSMA, CD20, CD105 (endoglin), ICAM-1, CD30, CD16A, CD38, MUC1, EGFR, KIR2DL1,2, NKG2A, tenascin-C, IGF (Insulin-like growth factor), CTLA-4, mesothelin, CD138, c-Met, Ang2, VEGF-A, CD79b, ENPD3, folate receptor α, TEM-1, GM2, glypican 3, macrophage inhibitory factor, CD74, Notch1, Notch2, Notch3, CD37, TLR-2, CD3, CSF-1R, FGFR2b, HLA-DR, GM-CSF, EphA3, B7-H3, CD123, gpA33, Frizzled7 receptor, DLL4, VEGF, RSPO, LIV-1, SLITRK6, Nectin-4, CD70, CD40, CD19, SEMA4D (CD100), CD25, MET, Tissue Factor, IL- 8, EGFR, cMet, KIR3DL2, Bst1 (CD157), P-cadherin, CEA, GITR, TAM (tumor associated macrophage), CEA, DLL4, Ang2, CD73, FGFR2, CXCR4, LAG-3, GITR, Fucosyl GM1, IGF -1, angiopoietin 2, CS
F-1R, FGFR3, OX40, BCMA, ErbB3, CD137 (4-1BB), PTK7, EFNA4, FAP, DR5, CEA, Ly6E, CA6, CEACAM5, LAMP1, tissue factor, EPHA2, DR5, B7-H3, FGFR4, FGFR2, α2-PI, A33, GDF15, CAIX, CD166, ROR1, GITR, BCMA, TBA, LAG-3, EphA2, TIM-3, CD-200, EGFRvIII, CD16A, CD32B, PIGF, Axl, MICA/B, Thomsen-Friedenreich, CD39, CD37, CD73, CLEC12A, Lgr3, Transferrin Receptor, TGFβ, IL-17, 5T4, RTK, Immune Suppressor Protein, NaPi2b, Lewis Blood Group B Antigen, A34, Lysil-Oxidase, DLK-1, TROP-2, α9 integrin, TAG-72 (CA72-4), CD70

(2) Autoimmune diseases/inflammatory diseases IL-17, IL-6R, IL-17R, INF-α, IL-5R, IL-13, IL-23, IL-6, ActRIIB, β7-Integrin, IL- 4αR, HAS, Eotaxin-1, CD3, CD19, TNF-α, IL-15, CD3ε, Fibronectin, IL-1β, IL-1α, IL-17, TSLP (Thymic Stromal Lymphopoietin), LAMP (Alpha4 Beta 7 Integrin) , IL-23, GM-CSFR, TSLP, CD28, CD40, TLR-3, BAFF-R, MAdCAM, IL-31R, IL-33, CD74, CD32B, CD79B, IgE (immunoglobulin E), IL-17A, IL-17F, C5, FcRn, CD28, TLR4, MCAM, B7RP1, CXCR1,2 Ligands, IL-21, Cadherin-11, CX3CL1, CCL20, IL-36R, IL-10R, CD86, TNF-α, IL-7R , Kv1.3, α9 integrin, LIFHT

(3) Cranial nerve disease CGRP, CD20, β-amyloid, β-amyloid protofibrin, Calcitonin Gene-Related Peptide Receptor, LINGO (Ig Domain Containing 1), α-synuclein, extracellular tau, CD52, insulin receptor, tau protein, TDP-43 , SOD1, TauC3, JC virus

(4) infectious diseases Clostridium difficile toxin B, cytomegalovirus, respiratory syncytial virus, LPS, S. Aureus Alpha-toxin, M2e protein, Psl, PcrV, S. Aureus toxin, Influenza A, Alginate, Staphylococcus aureus, PD-L1, Influenza B, Acinetobacter, F-protein, Env, CD3, Pathogenic Escherichia coli, Klebsiella, Streptococcus pneumoniae

(5) Hereditary/rare diseases Amyloid AL, SEMA4D (CD100), insulin receptor, ANGPTL3, IL4, IL13, FGF23, adrenocorticotropic hormone, transthyretin, huntingtin

(6) eye disease Factor D, IGF-1R, PGDFR, Ang2, VEGF-A, CD-105 (Endoglin), IGF-1R, β amyloid

(7) Bone/orthopedic Sclerostin, Myostatin, Dickkopf-1, GDF8, RNAKL, HAS, Siglec-15

(8) Blood diseases vWF, Factor IXa, Factor X, IFNγ, C5, BMP-6, Ferroportin, TFPI

(9) Other diseases BAFF (B cell activating factor), IL-1β, PCSK9, NGF, CD45, TLR-2, GLP-1, TNFR1, C5, CD40, LPA, prolactin receptor, VEGFR-1, CB1, Endoglin, PTH1R, CXCL1, CXCL8, IL-1β, AT2-R, IAPP

In a more preferred embodiment, the affinity agent for soluble proteins is an affinity agent for monoclonal antibodies. Isotypes for monoclonal antibodies are similar to those described above for antibodies, but IgG (eg, IgG1, IgG2, IgG3, IgG4) is preferred. Preferably, the monoclonal antibody is a full-length monoclonal antibody.

In an even more preferred embodiment, the affinity agent for a soluble protein is an affinity agent for a full-length monoclonal antibody, a chimeric antibody, a humanized antibody, or a human antibody (e.g., IgG such as IgG1, IgG2, IgG3, IgG4, etc.). .

In a particularly preferred embodiment, the affinity substance for a soluble protein is an affinity substance for an antibody that contains any one Fc region protein selected from the group consisting of (A) to (C) below and has antigen-binding ability. be:
(A) an Fc region protein comprising the amino acid sequence of SEQ ID NO: 1;
(B) an Fc region protein comprising an amino acid sequence in which one or several amino acid residues are inserted, added, deleted or substituted in the amino acid sequence of SEQ ID NO: 1; or (C) the amino acid sequence of SEQ ID NO: 1 and 90 An Fc region protein comprising an amino acid sequence exhibiting greater than or equal to % identity.

The amino acid sequence of SEQ ID NO: 1 is the Fc region protein. Such Fc region proteins are known to have secretory ability. Therefore, the Fc region proteins (A) to (C) above can have secretory ability. Antibodies comprising such Fc region proteins can also have antigen-binding ability. The amino acid residue at position 18 in SEQ ID NO: 1 is any amino acid residue, preferably a neutral amino acid residue, more preferably an amino acid residue having a non-polar side chain as described later, Leucine, isoleucine or alanine is more preferred, and leucine or alanine is particularly preferred. The amino acid residue at position 19 in SEQ ID NO: 1 is any amino acid residue, preferably a neutral amino acid residue or an acidic amino acid residue, more preferably an amino acid residue having a non-polar side chain or an acidic It is an amino acid residue, more preferably leucine or glutamic acid. The amino acid residue at position 21 in SEQ ID NO: 1 is any amino acid residue, preferably a neutral amino acid residue, more preferably an amino acid residue having a non-polar side chain, still more preferably glycine or alanine. The amino acid residue at position 140 in SEQ ID NO: 1 is any amino acid residue, preferably an acidic amino acid residue, more preferably glutamic acid or aspartic acid. The amino acid residue at position 142 in SEQ ID NO: 1 is any amino acid residue, preferably a neutral amino acid residue, more preferably an amino acid residue having a non-polar side chain, still more preferably Methionine, leucine or isoleucine, particularly preferably methionine or leucine. The amino acid residue at position 177 in SEQ ID NO: 1 is any amino acid residue, preferably a neutral amino acid residue, more preferably an amino acid residue having an uncharged polar side chain as described below, or It is an amino acid residue having a non-polar side chain, more preferably threonine, alanine or glycine, particularly preferably threonine or alanine.

In a preferred embodiment, the amino acid sequence of SEQ ID NO:1 may be an amino acid sequence consisting of amino acid residues at positions 220-449 in the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:41.

In another preferred embodiment, the amino acid sequence of SEQ ID NO:1 may be an amino acid sequence consisting of amino acid residues at positions 7-236 in the amino acid sequence of SEQ ID NO:3.

In certain embodiments, an antibody comprising an Fc region protein comprising an amino acid sequence as described above may be an antibody comprising an Fc region protein comprising an amino acid sequence as described above and an antibody constant region. The constant region of such an antibody may be a chimeric antibody, a humanized antibody, or a human antibody (eg, IgG such as IgG1, IgG2, IgG3, IgG4, etc.).

In the Fc region protein (B), one or several amino acid residues are modified by 1, 2, 3 or 4 mutations selected from the group consisting of deletion, substitution, addition and insertion of amino acid residues. obtain. Amino acid residue mutations may be introduced in one region in the amino acid sequence, but may also be introduced in a plurality of different regions. The term "one or several" indicates the number that does not significantly impair the activity of the protein. The number indicated by the term "one or several" is, for example, 1 to 100, preferably 1 to 80, more preferably 1 to 50, 1 to 30, 1 to 20, 1 to 10 or 1 to 5 (eg, 1, 2, 3, 4, or 5).

For the Fc region protein (C), the % identity with the amino acid sequence of SEQ ID NO: 1 is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% % or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. According to the present invention, the calculation of % identity of a peptide or polypeptide (protein) can be performed with the algorithm blastp. More specifically, the percentage identity of a polypeptide is calculated using the default Scoring Parameters (Matrix: BLOSUM62; Gap Costs: Existence=11 Extension = 1; Compositional Adjustments: Conditional compositional score matrix adjustment). Calculation of percent identity of polynucleotides (genes) can also be performed by the algorithm blastn. More specifically, the % identity of the polynucleotide is calculated using the default Scoring Parameters (Match/Mismatch Scores = 1, -2; Gap Costs = Linear) in the algorithm blastn provided in NCBI. It can be carried out.

Secretion in secretory capacity is synonymous with secretion of secretory proteins (so-called soluble). Therefore, "having secretory ability" means functioning as a soluble protein, like a normal antibody.

Antibodies comprising the Fc region protein may have mutations introduced at specific sites as long as the desired properties (eg, secretion ability, antigen-binding ability) are maintained. The positions of amino acid residues that may be mutated that may retain the properties of interest will be apparent to those skilled in the art. Specifically, one skilled in the art can 1) compare the amino acid sequences of multiple proteins with similar properties, 2) identify regions that are relatively conserved, and regions that are relatively non-conserved, and 3) since relatively conserved and relatively non-conserved regions can predict regions that may and may not play an important role in function, respectively. Recognize the relationship between structure and function. Therefore, those skilled in the art can identify the positions of amino acid residues that may be mutated in the amino acid sequence of the antibody containing the Fc region protein.

When amino acid residues are mutated by substitution, the substitution of amino acid residues may be conservative substitutions. As used herein, the term "conservative substitution" refers to the replacement of a given amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are well known in the art. For example, such families include amino acids with basic side chains (e.g. lysine, arginine, histidine), amino acids with acidic side chains (e.g. aspartic acid, glutamic acid), amino acids with uncharged polar side chains. (e.g. asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g. glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β branched side chains (e.g. threonine, valine, isoleucine), amino acids with aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine), amino acids with hydroxyl group (e.g. alcoholic, phenolic) containing side chains (e.g. serine, threonine, tyrosine), and amino acids with sulfur-containing side chains (eg, cysteine, methionine). Preferably, conservative amino acid substitutions are between aspartic acid and glutamic acid, between arginine and lysine and histidine, between tryptophan and phenylalanine, between phenylalanine and valine. , between leucine, isoleucine and alanine, and between glycine and alanine.

Antibodies comprising any one Fc region selected from the group consisting of (A) to (C) above include, for example, chimeric antibodies (e.g., rituximab, basiliximab, infliximab, cetuximab, siltuximab, dinutuximab, ortatoxasimab) , humanized antibodies (e.g., daclidumab, palivizumab, trastuzumab, alentuzumab, omalizumab, efalizumab, bevacizumab, natalizumab (IgG4), tocilizumab, eculizumab (IgG2), mogamulizumab, pertuzumab, obinutuzumab, vedolizumab, penprolizumab, erpolizumab (IgG4), daratumumab, ikesekizumab (IgG4), resulizumab (IgG4), atezolizumab), human antibodies (e.g., adalimumab, panitumumab, golimumab, ustekinumab, canakinumab, ofatumumab, denosumab (IgG2), ipilimumab, belimumab, ruxibakumab, ramucirumab, nivolumab (IgG4) secukinumab, evolocumab (IgG2), alirocumab, necitumumab, brodalumab (IgG2), olalatumab) (where no IgG subtype is mentioned, IgG1 is indicated).

Substances with affinity for soluble proteins as described above include, for example, peptides (including oligopeptides, polypeptides, and proteins), low-molecular-weight compounds, nucleic acids, nucleic acid-peptide complexes, peptide-low-molecular-weight compound complexes, nucleic acids - including low-molecular-weight complexes.

In certain embodiments, the affinity agent for a soluble protein as described above may be a peptide (including oligopeptides, polypeptides, proteins, which may be glycoproteins). For example, the following peptides have been reported:
(1) human IgG in general (ie, human IgG1, IgG2, IgG3 and IgG4;
IgG-binding peptides (e.g., WO 2016/186206, WO 2013/027796, see WO 2008/054030) that have affinity for a specific region (CH2 region) of hereinafter the same);
(2) Protein A mimetic (PAM) peptide having affinity for a specific region (CH2 region) of human IgG in general (see, for example, Fassina G et al., JOURNAL OF MOLECULAR RECOGNIZATION, 1996, VOL.6, 564-569) ;
(3) EPIHRSTTALL (SEQ ID NO: 9) having affinity for a specific region (CH2 region) of general human IgG (e.g., Ehrlich GK et al., J. Biochem. Biophys. Methods, 2001, VOL. 49, 443 -454);
(4) (NH2-Cys1-X1-X2-X3-X4) 2-Lys-Gly-OH (e.g., Ruvo M et al., ChemBioChem, 2005) that has affinity for a specific region (Fc region) of human IgG in general , VOL.6, 1242-1253);
(5) FARLVSSIRY (SEQ ID NO: 10), FGRLVSSIRY (SEQ ID NO: 11), and TWKTSRISIF (SEQ ID NO: 12) having affinity for specific regions (Fc regions) of general human IgG (eg, Krook M et al., Journal of Immunological Methods, 1998, VOL.221, 151-157);
(6) QSYP (SEQ ID NO: 13) having affinity for specific regions of human IgG in general (see, for example, Jacobs JM et al., Bio.Techniques, 2003, VOL.34, 132-141);
(7) HWRGWV (SEQ ID NO: 14), HYFKFD (SEQ ID NO: 15), and HFRRHL (SEQ ID NO: 16) having affinity for a specific region (Fc region) of general human IgG (eg, Carbonell RG et al. , Journal of Chromatography A, 2009, VOL.1216, 910-918);
(8) DAAG (SEQ ID NO: 17) having affinity for a specific region (Fc region) of general human IgG (e.g., Lund LN et al., Journal of Chromatography A, 2012, VOL.1225, 158-167 );
(9) Fc-I, Fc-II, and Fc-III having affinity for a specific region (Fc region) of general human IgG (e.g., Warren L. Delano et al., Science, 2000, VOL.287, 1279 -1283; see WO 2001/045746); and (10) NARKFYKG (SEQ ID NO: 18) and NKFRGKYK (SEQ ID NO: 19) with affinity to specific regions (Fc regions) of human IgG in general (e.g., Biochemical
Engineering Journal, 2013, VOL. 79, 33-40).

In another particular embodiment, the affinity substance for soluble proteins as described above may be substances other than peptides. Such substances include, for example, aptamers having affinity for specific regions (CH2 region, especially the side chain of Lys340) of human IgG (e.g., human IgG1-4) [e.g., GGUGs such as GGUGCU and GGUGAU (C/ A) (U / T) motif-containing aptamers] have been reported (e.g., International Publication No. 2007/004748; Nomura Y et al., Nucleic Acids Res., 2010 Nov; 38(21): 7822-9 Miyakawa S et al., RNA., 2008 Jun;14(6):1154-63).

Affinity substances for soluble proteins as described above can be obtained by any known method in the art. For example, by using a whole soluble protein or a partial peptide in the soluble protein (e.g., when the surface-exposed region of the protein is known, a partial peptide present in the region) to produce an antibody (e.g., hybridoma method), or by screening affinity substances from libraries where affinity substances are available (e.g., peptide libraries, antibody libraries, antibody-producing cell libraries, aptamer libraries, phage libraries, mRNA libraries, cDNA libraries) (e.g., phage display method, SELEX method, mRNA display method, ribosome display method, cDNA display method, yeast display method). Further, when the substance with affinity for soluble proteins is a substance with affinity for the Fc region (soluble region) of an antibody, a specific region (eg, CH1 , CH2, CH3), an affinity substance (e.g., antibody, aptamer) capable of selectively binding to any portion in the Fc region of an antibody can be efficiently produced. can be obtained. Among the affinity substances obtained in this way, there is a mixture of those with relatively strong and weak affinity binding abilities. However, even an affinity substance with weak affinity binding ability can be used in an excessive amount to reinforce the affinity binding ability.

The affinity substance for soluble proteins as described above may be an IgG-binding peptide represented by the following formula (i) or a salt thereof (eg, Examples and WO 2016/186206).
(X _1-3 )-C-(X ₂ )-H-(Xaa1)-G-(Xaa2)-LVWC-(X _1-3 ) (SEQ ID NO: 94) (i)
[In the formula,
X is the same or different and is any amino acid residue other than cysteine;
C is a cysteine residue,
H is a histidine residue,
Xaa1 is an arginine residue, a leucine residue, a lysine residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue;
G is a glycine residue,
Xaa2 is a lysine residue, a glutamine residue, a glutamic acid residue, an asparagine residue, or an aspartic acid residue;
L is a leucine residue,
V is a valine residue and W is a tryptophan residue. ]
A peptide or a salt thereof comprising an amino acid sequence consisting of 13 to 17 amino acid residues represented by and capable of binding to human IgG and/or rabbit IgG.
Xaa1 and Xaa2 are preferably different amino acid residues.

As used herein, the notation X _1-3 at the N-terminus or C-terminus means that 1 to 3 consecutive arbitrary amino acid residues X independently other than cysteine (C or Cys), The amino acid residues that constitute it are the same or different residues, but preferably consist of a sequence in which all three are not the same residues. Similarly, X2 also means that two consecutive arbitrary amino acid residues X other than cysteine (C or Cys) are consecutive, and the amino acid residues constituting it are the same or different residues. However, preferably the two consecutive amino acid residues consist of a sequence that is not the same residue. X1, which will be described later, also means that there is one independently arbitrary amino acid residue X other than cysteine (C or Cys).

Also herein, at least two cysteine residues spaced in each amino acid sequence of the peptide can form a cyclic peptide through disulfide bonds. Generally, in peptides of formulas such as formula (i) above, the outer two cysteine residues are disulfide-bonded. Alternatively, in the peptide of the formula (i) above, the sulfide groups in the two outer cysteine residues may be linked by a carbonyl group-containing linker shown below.

The dashed portion of the carbonyl group-containing linker represented above means the portion that binds to the sulfide group. The linker is more stable against reduction reactions and the like than ordinary disulfide bonds. This peptide can be prepared, for example, by the method described in WO2016/186206.

In certain embodiments, the affinity substance of formula (i) above may be an IgG-binding peptide represented by formula (i-1) below or a salt thereof (eg, International Publication No. 2016/186206 ).
(X _1-3 )-C-(X ₂ )-H-(Xaa1)-G-(Xaa2)-LVWC-(X _1-3 ) (SEQ ID NO: 95) (i-1)
[In the formula,
X is the same or different and is any amino acid residue other than cysteine;
C is a cysteine residue,
H is a histidine residue,
Xaa1 is a lysine residue, a cysteine residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue;
G is a glycine residue,
Xaa2 is a glutamic acid residue or an asparagine residue,
L is a leucine residue,
V is a valine residue and W is a tryptophan residue. ]
A peptide or a salt thereof comprising an amino acid sequence consisting of 13 to 17 amino acid residues represented by and capable of binding to human IgG and/or rabbit IgG.
Xaa1 and Xaa2 are preferably different amino acid residues.

In another specific embodiment, the affinity substance for soluble proteins as described above may be an IgG-binding peptide represented by the following formula (i-2) or a salt thereof (eg, Examples).
(X _1-3 )-C-(X ₂ )-H-(Xaa1)-G-(Xaa2)-LVWC-(X _1-3 ) (SEQ ID NO: 96) (i-2)
[In the formula,
X is the same or different and is any amino acid residue other than cysteine;
C is a cysteine residue,
H is a histidine residue,
Xaa1 is an arginine residue or a leucine residue,
G is a glycine residue,
Xaa2 is a lysine residue, a glutamine residue, or an aspartic acid residue;
L is a leucine residue,
V is a valine residue and W is a tryptophan residue. ] and is capable of binding to human IgG and/or rabbit IgG, or a salt thereof.
The affinity substance not disclosed in WO 2016/186206 having such a specific structure is Lys248 or Lys246 according to Eu numbering in human IgG Fc, or other than Lys248 or Lys246 (Example).

Peptides represented by formula (i-1′) and formula (i-1″) in which amino acid residue X is further specified in the amino acid sequence of the peptide of formula (i-1) are shown below.

That is, the peptide represented by formula (i-1') is
(X _1-3 )-C-(X1)-YH-(Xaa1)-GNLVWC-(X _1-3 ) (SEQ ID NO: 97) (i-1′)
[In the formula,
X is the same or different and is any amino acid residue other than cysteine;
C is a cysteine residue,
Y is a tyrosine residue;
H is a histidine residue,
Xaa1 is a lysine residue, a cysteine residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue;
G is a glycine residue,
N is an asparagine residue,
L is a leucine residue,
V is a valine residue and W is a tryptophan residue. ]
and is capable of binding to human IgG and/or rabbit IgG.

The peptide represented by formula (i-1'') is
(X _1-3 )-CA-(X1)-H-(Xaa1)-GELVWC-(X _1-3 ) (SEQ ID NO: 98) (i-1″ )
[In the formula,
X is the same or different and is any amino acid residue other than cysteine;
C is a cysteine residue,
A is an alanine residue,
H is a histidine residue,
Xaa1 is a lysine residue, a cysteine residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue;
G is a glycine residue,
E is a glutamic acid residue,
L is a leucine residue,
V is a valine residue and W is a tryptophan residue. ]
and is capable of binding to human IgG and/or rabbit IgG.

A peptide represented by formula (ii), in which amino acid residue X is further specified in the amino acid sequence of the peptide of formula (i-1), is shown below.

That is, the peptide represented by formula (ii) is
(X _1-3 )-C-(Xaa3)-(Xaa4)-H-(Xaa1)-G-(Xaa2)
-LVWC-(X _1-3 ) (SEQ ID NO: 99) (ii)
[In the formula,
X is the same or different and is any amino acid residue other than cysteine;
C is a cysteine residue,
H is a histidine residue,
Xaa1 is a lysine residue, a cysteine residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue;
G is a glycine residue,
Xaa2 is a glutamic acid residue or an asparagine residue;
L is a leucine residue,
V is a valine residue,
W is a tryptophan residue,
Xaa3 is an alanine, serine or threonine residue, and Xaa4 is a tyrosine or tryptophan residue. ]
and is capable of binding to human IgG and/or rabbit IgG.

In the amino acid sequence of the peptide of the formula such as formula (i), when 17 amino acid residues are used, the 1st and 2nd, and 16th and 17th amino acid residues X from the N-terminus may be deleted. , such a peptide consists of 13 amino acids in length.

As used herein, the phrase "assuming 17 amino acid residues" refers to N of 17 residues, which is the longest amino acid length for peptides such as those of formula (i), when the amino acid residues of the peptide are referred to by amino acid number. It is a term used for convenience in order to number from 1st to 17th in order from the end.

A peptide represented by formula (iii), in which amino acid residue X is further specified in the amino acid sequence of the peptide of formula (i-1), is shown below.

That is, the peptide represented by formula (iii) is
(X _1-3 )-CAYH-(Xaa1)-GEL-VWC-(X _1-3 )
(SEQ ID NO: 100) (iii)
[In the formula,
X is the same or different and is any amino acid residue other than cysteine;
C is a cysteine residue,
A is an alanine residue,
Y is a tyrosine residue;
H is a histidine residue,
Xaa1 is a lysine residue, a cysteine residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue;
G is a glycine residue,
E is a glutamic acid residue;
L is a leucine residue,
V is a valine residue and W is a tryptophan residue. ]
and is capable of binding to human IgG and/or rabbit IgG.

In the amino acid sequence of the peptide of formula (iii), when 17 amino acid residues are used, the 1st and 2nd amino acid residues and the 16th and 17th amino acid residues X from the N-terminus may be deleted. A typical peptide consists of 13 amino acids in length.

Furthermore, amino acid residues other than cysteine (C) in the amino acid sequence of the peptides of the above formulas, that is, when 17 amino acid residues, the 1st to 3rd, 5th, 6th, 15th to 17th amino acids from the N-terminus Preferably, residues are selected from: where each capital letter is the single-letter code for an amino acid:
1st amino acid residue = S, G, F or none 2nd amino acid residue = D, G, A, S, P, homocysteine or none 3rd amino acid residue = S, D, T, N, E or R,
15th amino acid residue = S, T or D,
16th amino acid residue = H, G, Y, T, N, D, F, homocysteine or none;
17th amino acid residue = Y, F, H, M or none.
5th amino acid residue = A or T,
6th amino acid residue = Y or W;

A peptide represented by formula (iv), in which amino acid residue X is further specified in the amino acid sequence of the peptide of formula (i-1), is shown below.

That is, the peptide represented by formula (iv) is
DC-(Xaa3)-(Xaa4)-H-(Xaa1)-G-(Xaa2)-LVWCT (SEQ ID NO: 101) (iv)
[In the formula,
D is an aspartic acid residue,
C is a cysteine residue,
H is a histidine residue,
Xaa1 is a lysine residue, a cysteine residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue;
G is a glycine residue,
Xaa2 is a glutamic acid residue or an asparagine residue;
L is a leucine residue,
V is a valine residue,
W is a tryptophan residue,
T is a threonine residue,
Xaa3 is an alanine residue or a threonine residue, and
Xaa4 is a tyrosine or tryptophan residue. ] and is capable of binding to human IgG and/or rabbit IgG.

Some specific examples of peptides of formula (i-1) are listed in (1) to (19) below, but needless to say, they are not limited to:
(1) DCAYH(Xaa1)GELVWCT (SEQ ID NO: 20);
(2) GPDCAYH (Xaa1) GELVWCTFH (SEQ ID NO: 21);
(3) RCAYH(Xaa1)GELVWCS (SEQ ID NO: 22);
(4) GPRCAYH(Xaa1)GELVWCSFH (SEQ ID NO: 23);
(5) SPDCAYH (Xaa1)GELVWCTFH (SEQ ID NO: 24);
(6) GDDCAYH (Xaa1) GELVWCTFH (SEQ ID NO: 25);
(7) GPSCAYH(Xaa1)GELVWCTFH (SEQ ID NO: 26);
(8) GPDCAYH (Xaa1) GELVWCSFH (SEQ ID NO: 27);
(9) GPDCAYH (Xaa1) GELVWCTHH (SEQ ID NO: 28);
(10) GPDCAYH(Xaa1)GELVWCTFY (SEQ ID NO: 29);
(11) SPDCAYH(Xaa1)GELVWCTFY (SEQ ID NO: 30);
(12) SDDCAYH(Xaa1)GELVWCTFY (SEQ ID NO: 31);
(13) RGNCAYH (Xaa1) GQLVWCTYH (SEQ ID NO: 32);
(14) G(Xaa2)DCAYH(Xaa1)GELVWCT(Xaa2)H (SEQ ID NO: 33);
(15) RRGPDCAYH(Xaa1)GELVWCTFH (SEQ ID NO: 34);
(16) DCTYH(Xaa1)GNLVWCT (SEQ ID NO: 35);
(17) DCAYH(Xaa1)GNLVWCT (SEQ ID NO: 36);
(18) DCTYH(Xaa1)GELVWCT (SEQ ID NO:37); and (19) DCAWH(Xaa1)GELVWCT (SEQ ID NO:38).
[In the formula,
Xaa1 is a lysine residue, a cysteine residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue;
Xaa2 is homocysteine;
Preferably, the homocysteines form disulfide bonds with each other. ].

Preferable specific examples of the peptide of formula (i-1) include the following:
(1) DCAYH(Xaa1)GELVWCT (SEQ ID NO: 20);
(2) GPDCAYH (Xaa1) GELVWCTFH (SEQ ID NO: 21);
(13) RGNCAYH (Xaa1) GQLVWCTYH (SEQ ID NO: 22);
(14) G(Xaa2)DCAYH(Xaa1)GELVWCT(Xaa2)H (SEQ ID NO:33); and (15) RRGPDCAYH(Xaa1)GELVWCTFH (SEQ ID NO:34).
[In the formula,
Xaa1 is a lysine residue,
Xaa2 is homocysteine;
Preferably, cysteines and/or homocysteines form disulfide bonds with each other. ]

The peptide of (13) above may be RGNCAYHKGQLVWCTYH (SEQ ID NO: 39).

In another specific embodiment, the affinity substance of formula (i) above may be an IgG-binding peptide represented by formula (v) below or a salt thereof (eg, Examples).
(X _1-3 )-C-(Xaa3)-(xaa4)-H-(Xaa1)-G-(Xaa2)-LVWC-(Xaa5)-(Xaa6)-(Xaa7) (sequence number 102)
(v)
[In the formula,
X is the same or different and is any amino acid residue other than cysteine;
C is a cysteine residue,
Xaa3 is an alanine residue or a lysine residue,
Xaa4 is a tryptophan residue or a tyrosine residue,
H is a histidine residue,
Xaa1 is an arginine residue, a leucine residue, a lysine residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue;
G is a glycine residue,
Xaa2 is a lysine residue, a glutamine residue, a glutamic acid residue, an asparagine residue, or an aspartic acid residue;
L is a leucine residue,
V is a valine residue,
W is a tryptophan residue,
Xaa5 is a threonine residue or a lysine residue,
Xaa6 is a tyrosine residue, a lysine residue, or no residue, and Xaa7 is a histidine residue, a lysine residue, or no residue. ] and is capable of binding to human IgG, or a salt thereof.

The affinity substance not disclosed in WO 2016/186206 having such a specific structure is Lys248 or Lys246 according to Eu numbering in human IgG Fc, or other than Lys248 or Lys246 (Example). Preferably, any one of Xaa3, Xaa1, Xaa2, Xaa5, Xaa6 and Xaa7 is a lysine residue. Xaa1 may preferably be an arginine residue or a leucine residue. Alternatively, Xaa1 is preferably a lysine residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue, more preferably a lysine residue or an aspartic acid residue. , or glutamic acid residues.

In the amino acid sequence of the peptide of formula (v), when 17 amino acid residues are used, the 1st and 2nd, and 16th and 17th amino acid residues X from the N-terminus may be deleted. A typical peptide consists of 13 amino acids in length.

Furthermore, when amino acid residues other than cysteine (C) in the amino acid sequence of the peptide of the above formula (v), that is, 17 amino acid residues, each of the 1st to 3rd amino acid residues from the N-terminus has the following: is preferably selected from where each capital letter is the single-letter code for an amino acid:
1st amino acid residue = R, S, G, F or none (preferably R or none)
2nd amino acid residue = D, G, A, S, P, homocysteine or none (preferably G or none)
3rd amino acid residue = S, D, T, N, E or R (preferably N or D).

Some specific examples of peptides of formula (v) are listed in (16) to (34) below, but are of course not limited to:
(16) RGNCAYH(Xaa1)GQLVWCTYH (SEQ ID NO: 73)
(17) RGNCAWH(Xaa1)GQLVWCTYH (SEQ ID NO: 74)
(18) RGNCAWH(Xaa1)GELVWCTYH (SEQ ID NO: 75)
(19) RGNCKWH(Xaa1)GQLVWCTYH (SEQ ID NO: 76)
(20) RGNCKYH (Xaa1) GELVWCTYH (SEQ ID NO: 77)
(21) RGNCKYH(Xaa1)GQLVWCTYH (SEQ ID NO: 78)
(22) DCKWH(Xaa1)GELVWCT (SEQ ID NO: 79)
(23) DCKYH(Xaa1)GELVWCT (SEQ ID NO: 80)
(24) DCKWH(Xaa1)GELVWCT (SEQ ID NO: 81)
(25) DCKWH(Xaa1)GQLVWCT (SEQ ID NO: 82)
(26) DCKYH(Xaa1)GELVWCT (SEQ ID NO: 83)
(27) DCKYH(Xaa1)GQLVWCT (SEQ ID NO: 84)
(28) DCKWH(Xaa1)GQLVWCT (SEQ ID NO: 85)
(29) DCKYH(Xaa1)GQLVWCT (SEQ ID NO: 86)
(30) RGNCAWH(Xaa1)GQLVWCKYH (SEQ ID NO: 87)
(31) RGNCAWH(Xaa1)GELVWCKYH (SEQ ID NO: 88)
(32) RGNCAYH(Xaa1)GQLVWCTKH (SEQ ID NO: 89)
(33) RGNCAYH(Xaa1)GQLVWCTYK (SEQ ID NO: 90)
(34) RGNCAYH (Xaa1) GQLVWCTKH (SEQ ID NO: 91).
[In the formula,
Xaa1 is an arginine residue, a leucine residue, a lysine residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue. ].
Xaa1 is preferably an arginine residue, a leucine residue or a lysine residue, more preferably a lysine residue.

Further, the IgG-binding peptide has the following formula (vi) as a broad primary structure:
DC-(Xaa3)-(Xaa4)-H-(Xaa1)-G-(Xaa2)-LVWC-(Xaa5)-(Xaa6)-(Xaa7) (SEQ ID NO: 103) (vi ) [In the formula,
D is an aspartic acid residue,
C is a cysteine residue,
Xaa3 is an alanine residue or a lysine residue,
Xaa4 is a tryptophan residue or a tyrosine residue,
H is a histidine residue,
Xaa1 is an arginine residue, a leucine residue, a lysine residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue;
G is a glycine residue,
Xaa2 is a lysine residue, a glutamine residue, a glutamic acid residue, an asparagine residue, or an aspartic acid residue;
L is a leucine residue,
V is a valine residue,
W is a tryptophan residue,
Xaa5 is a threonine residue or a lysine residue,
Xaa6 is a tyrosine residue, a lysine residue, or no residue;
Xaa7 is a histidine residue, a lysine residue, or none. ] and is capable of binding to human IgG and/or rabbit IgG (e.g., Examples and International Publication No. 2016 /186206).
Preferably, any one of Xaa3, Xaa1, Xaa2, Xaa5, Xaa6 and Xaa7 is a lysine residue. Xaa1 is preferably a lysine, arginine, or leucine residue, and Xaa2 is preferably a lysine, glutamine, or glutamic acid residue. .

In certain embodiments, the IgG binding peptide has formula (vii) below:
DC-(Xaa3)-(Xaa4)-H-(Xaa1)-G-(Xaa2)-LVWCT (SEQ ID NO: 104) (vii)
[In the formula,
D is an aspartic acid residue,
C is a cysteine residue,
Xaa3 is an alanine residue or a lysine residue,
Xaa4 is a tryptophan residue or a tyrosine residue,
H is a histidine residue,
Xaa1 is an arginine residue, a leucine residue, a lysine residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue;
G is a glycine residue,
Xaa2 is a lysine residue, a glutamine residue, a glutamic acid residue, an asparagine residue, or an aspartic acid residue;
L is a leucine residue,
V is a valine residue,
W is a tryptophan residue and T is a threonine residue. ] and is capable of binding to human IgG and/or rabbit IgG (eg, International Publication No. 2016/186206). Preferably, any one of Xaa3, Xaa1 and Xaa2 is a lysine residue. Xaa1 is preferably a lysine, arginine, or leucine residue, and Xaa2 is preferably a lysine, glutamine, or glutamic acid residue.

In another specific embodiment, the IgG binding peptide has formula (viii) below:
RGNC-(Xaa3)-(Xaa4)-H-(Xaa1)-G-(Xaa2)-LVWC-(Xaa5)-(Xaa6)-(Xaa7) (SEQ ID NO: 105) (viii)
[In the formula,
R is an arginine residue,
G is a glycine residue,
N is an asparagine residue,
C is a cysteine residue,
Xaa3 is an alanine residue or a lysine residue,
Xaa4 is a tryptophan residue or a tyrosine residue,
H is a histidine residue,
Xaa1 is an arginine residue, a leucine residue, a lysine residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue;
G is a glycine residue,
Xaa2 is a lysine residue, a glutamine residue, a glutamic acid residue, an asparagine residue, or an aspartic acid residue;
L is a leucine residue,
V is a valine residue,
W is a tryptophan residue,
Xaa5 is a threonine residue or a lysine residue,
Xaa6 is a tyrosine residue, a lysine residue, or no residue, and Xaa7 is a histidine residue, a lysine residue, or no residue. ] and is capable of binding to human IgG and/or rabbit IgG (eg, Examples). Compounds not disclosed in WO 2016/186206 having such a specific structure are Lys248 or Lys246 residues according to Eu numbering in human IgG Fc, or other amino acids other than Lys248 or Lys246 residues Useful for regioselective modification of residues (Examples). Preferably, any one of Xaa3, Xaa1, Xaa2, Xaa5, Xaa6 and Xaa7 is a lysine residue. Xaa1 may preferably be an arginine residue or a leucine residue. Alternatively, Xaa1 is preferably a lysine, arginine or leucine residue, and Xaa2 is preferably a lysine, glutamine or glutamic acid residue.

The peptide has at least two cysteine (C) residues spaced apart in each amino acid sequence disulfide-bonded to form a cyclic peptide, with one or two cysteine (C) residues at the N-terminal and C-terminal sides of each cysteine residue. It may have any amino acid residue other than cysteine. When each cysteine residue has 1 or 2 amino acid residues on the N-terminal side and the C-terminal side, and 17 amino acid residues, each of the 1st to 2nd and 16th to 17th amino acid residues from the N-terminus The groups are those exemplified above. In addition, the amino acids constituting the peptide may be either L-form or D-form, but the L-form is preferred (in the Examples, all the amino acid residues forming the peptide are L-form).

As described above, in the IgG-binding peptide, the Xaa amino acid residue can be easily modified with a cross-linking agent (lysine residue, cysteine residue, aspartic acid residue, or protein-constituting amino acid residue such as glutamic acid residue). , or non-proteinogenic amino acids such as diaminopropionic acid residues or 2-aminosuberic acid residues), among these amino acids, lysine residues are preferred. Examples of such cross-linking agents include cross-linking agents containing preferably two or more succinimidyl groups such as DSG (disuccinimidyl glutarate), DSS (disuccinimidyl suberate), and DMA ( dimethyl adipimidate.2HCl, dimethyl adipimidate dihydrochloride), DMP (dimethyl pimelimidate.2 HCl, dimethylpimelimidate dihydrochloride), and DMS (dimethyl suberimidate.2
and DTBP (dimethyl 3,3′-dithiobispropionimidate 2HCl, 3,3′-dithiobispropionimidate dimethyl dihydrochloride) ) and DSP (dithiobis (succinimidyl propionate), dithiobissuccinimidyl propionate) and other cross-linking agents having SS bonds (eg, International Publication No. 2016/186206). To increase site specificity in modifying the IgG-binding peptide with a cross-linking agent, the IgG-binding peptide has no or few residues in its sequence that are the same as Xaa1 (e.g., 1 or only two). For example, if Xaa1 is a lysine residue, the IgG-binding peptide preferably has no or few lysine residues in its sequence other than Xaa1.

IgG binding peptides bind to the Fc domain of IgG. The IgG-binding peptide is close to a specific region of IgG Fc, that is, Lys248 or Lys246 according to Eu numbering in human IgG Fc, preferably Lys248, in the amino acid residues of Xaa such as Xaa1 (implementation see Examples and WO2016/186206). Alternatively, in the IgG-binding peptide, the Xaa amino acid residue can be adjacent to other amino acid residues other than the Lys248 or Lys246 residue according to Eu numbering in human IgG Fc.

More specifically, the peptides represented by formulas (i) to (viii) above are as follows.
(1′) RGNCAYHKGQLVWCTYH (SEQ ID NO: 39)
(2′) RGNCKYHRGQLVWCTYH (SEQ ID NO: 42)
(3′) RGNCAWHRGKLVWCTYH (SEQ ID NO: 43)
(4′) RGNCKWHRGELVWCTYH (SEQ ID NO: 44)
(5′) RGNCKWHRGQLVWCTYH (SEQ ID NO: 45)
(6′) RGNCKYHLGELVWCTYH (SEQ ID NO: 46)
(7′) RGNCKYHLGQLVWCTYH (SEQ ID NO: 47)
(8′) DCKWHLGELVWCT (SEQ ID NO: 48)
(9') DCKYHLGELVWCT (SEQ ID NO: 49)
(10') DCKWHRGELVWCT (SEQ ID NO: 50)
(11′) DCKWHLGQLVWCT (SEQ ID NO: 51)
(12') DCKYHRGELVWCT (SEQ ID NO: 52)
(13') DCKYHLGQLVWCT (SEQ ID NO: 53)
(14') DCKWHRGQLVWCT (SEQ ID NO: 54)
(15') DCKYHRGQLVWCT (SEQ ID NO: 55)
(16') RGNCAWHLGQLVWCKYH (SEQ ID NO: 56)
(17') RGNCAWHLGELVWCKYH (SEQ ID NO: 57)
(18′) RGNCAYHLGQLVWCTKH (SEQ ID NO: 58)
(19′) RGNCAYHLGQLVWCTYK (SEQ ID NO: 59)
(20') RGNCAYHRGQLVWCTKH (SEQ ID NO: 60)

In addition, the affinity substance for soluble proteins as described above,
(a) in the amino acid sequence of FNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 92), any amino acid residue is a lysine residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, and a diaminopropionic acid residue ( amino acid residue that can be easily modified by a cross-linking agent) (preferably a lysine residue, an aspartic acid residue, or a glutamic acid residue, more preferably a lysine residue) and (b) an amino acid sequence having 90% or more identity to said amino acid sequence of SEQ ID NO:92.

Preferably, said amino acid sequences having properties (a) and (b) are capable of binding human IgG as described herein.

A peptide consisting of the amino acid sequence of SEQ ID NO: 92 is an affinity peptide known as Z34C in which two K (lysine) at positions 26 and 28 counted from the N-terminus are replaced with R (arginine) for the convenience of peptide reagent synthesis. ), which can be used by acetylating the N-terminus and amidating the C-terminus. More specifically, such affinity peptides include Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC-NH ₂ (SEQ ID NO:92). Affinity substances having such a specific structure are Lys248 or Lys246 residues according to Eu numbering in human IgG Fc, Lys288 or Lys290, Lys317, or other amino acid residues other than those residues. Useful for modification (Examples). The amino acid sequence of Z34C is FNMQCQRRFYEALHDPNLNEEQRNAKIKSIRDDC (SEQ ID NO: 93) (eg, Starovasnik, M.A. et al., Structural mimicry of an native protein by a minimized protein binding, Nas binding. USA., 94, 10080-10085 (1997)).

The affinity peptide can have affinity for human IgG (eg, human IgG as described above, preferably human IgG1). The affinity peptide may form a cyclic peptide by disulfide bonding of cysteine residues at positions 5 and 34.

Any position can be used as the position into which an amino acid residue that can be easily modified with a cross-linking agent is introduced, as long as it has affinity for human IgG such as human IgG1. Such positions can be readily identified by those skilled in the art. Preferably, the position where the amino acid residue that can be easily modified by a cross-linking agent is introduced is an amino acid residue other than the cysteine residues at positions 5 and 34, which may be disulfide-bonded. More preferably, the positions into which amino acid residues that can be easily modified by a cross-linking agent are introduced are, for example, positions 1, 3, 6, 7, 13, 20, 24, 31 and 32. position amino acid residues.

Preferably, said amino acid sequence having the properties of (a) and (b) comprises a lysine residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue and a diaminopropionic acid residue ( one specific amino acid residue (preferably a lysine residue, an aspartic acid residue, or a glutamic acid residue, more preferably a lysine residue) selected from the group consisting of easily modifiable amino acid residues) and 20 normal amino acid residues that constitute natural proteins (preferably 17 amino acid residues other than lysine residues, aspartic acid residues, and glutamic acid residues, and more It is also preferred to have mutations of 19 amino acid residues (preferably 19 amino acid residues other than lysine residues) at positions other than the predetermined positions. Such predetermined positions are not particularly limited, and include, for example, 1st, 3rd, 6th, 7th, 13th, 20th, 24th, 31st and 32nd positions. The amino acid sequence having the characteristics of (a) and (b) maintains two cysteine residues at positions 5 and 34, and these two cysteine residues may be linked by disulfide bonds. . The amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 92 has 1, 2, 3 or 4 mutations selected from the group consisting of deletion, substitution, addition and insertion of amino acid residues. 1 to 3 (preferably 1 or 2, more preferably 1) amino acid residues may be modified (preferably by substitution). Amino acid residue mutations may be introduced in one region in the amino acid sequence, but may also be introduced in a plurality of different regions.

More preferably, the above amino acid sequence having properties (a) and (b) may be (c) or (d) below.
(c) an amino acid sequence selected from the group consisting of the following amino acid sequences (1) to (9):
(1) KNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 61);
(2) FNMQCQKRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 62);
(3) FNMQCQRRFYEAKHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 63);
(4) FNMQCQRRFYEALHDPNLNEEQRKARIRSIRDDC (SEQ ID NO: 64);
(5) FNMQCQRRFYEALHDPNLNKEQRNARIRSIRDDC (SEQ ID NO: 65);
(6) FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 68);
(7) FNKQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 70);
(8) FNMQCKRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 71); and (9) FNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 72); or (d) one lysine residue and two cysteines in the amino acid sequence of any of (1) to (9) above. 19 amino acid residues other than lysine residues at positions other than residues (e.g., positions 1, 3, 6, 7, 13, 20, 24, 31, and 32) Amino acids having 90% or more identity to the amino acid sequence of any one of (1) to (9) above (which may be modifications of the number of amino acid residues as described above), which have mutations in groups arrangement. The above amino acid sequence of (d) preferably maintains two cysteine residues at positions 5 and 34, and these two cysteine residues may be linked by a disulfide bond. Preferably, the affinity peptide having the above amino acid sequence of (d) is capable of binding human IgG as described herein.

As long as the affinity peptide has 90% or more identity to the amino acid sequence of SEQ ID NO: 92 or the amino acid sequences of (1) to (9) above, one amino acid residue that can be easily modified with a cross-linking agent. In addition to the introduction of the group, it may have additional amino acid residue mutations. Positions at which additional amino acid mutations can be introduced can be readily identified by those skilled in the art. As such positions, for example, positions other than the 5- and 34-position cysteine residues can be used. For example, a phenylalanine residue at position 1, an arginine residue at position 6, a leucine residue at position 13, a glutamic acid residue at position 20, an asparagine residue at position 24, or an arginine residue at position 31 (which can be easily excluding positions where modifiable amino acid residues have already been introduced). Examples of amino acids that can be introduced by further amino acid mutation include alanine (A), asparagine (N), cysteine (C), glutamine (Q), glycine (G), isoleucine (I), leucine (L), methionine ( M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), valine (V), aspartic acid (D), glutamic acid (E), arginine (R), histidine (H), and lysine (L). Preferably, these 19 amino acids other than lysine may be utilized. Amino acids may be in either the L-form or the D-form, but the L-form is preferred (in the Examples, all the amino acid residues constituting the peptide are in the L-form).

The degree of % identity to said amino acid sequence of SEQ ID NO: 92 or said amino acid sequences of (1)-(9) can be determined as described above. The percent identity is preferably 92% or higher, more preferably 94% or higher, even more preferably 95% or higher, and particularly preferably 97% or higher (that is, with respect to the amino acid sequence of SEQ ID NO: 92, the lysine residue , aspartic acid residue, glutamic acid residue, 2-aminosuberic acid residue, and diaminopropionic acid residue).

When the affinity substance is a peptide, the terminal amino group and carboxy group of the peptide may be protected. Examples of protective groups for the N-terminal amino group include alkylcarbonyl groups (acyl groups) (eg, acetyl, propoxy, butoxycarbonyl groups such as tert-butoxycarbonyl groups), alkyloxycarbonyl groups (eg, fluorescein nylmethoxycarbonyl group), aryloxycarbonyl group, arylalkyl(aralkyl)oxycarbonyl group (eg, benzyloxycarbonyl group). An acetyl group is preferred as the protective group for the N-terminal amino group. Protecting groups for the C-terminal carboxy group include, for example, groups capable of forming esters or amides. Groups capable of forming esters or amides include, for example, alkyloxy groups (e.g., methyloxy, ethyloxy, propyloxy, butyloxy, pentyloxy, hexyloxy), aryloxy groups (e.g., phenyloxy, naphthyloxy), aralkyl An oxy group (eg, benzyloxy) and an amino group can be mentioned. As a protective group for the C-terminal carboxyl group, an amino group is preferred. When the affinity substance is a peptide containing two or more cysteine residues, disulfide bonds may be formed via thiol groups on the side chains of the cysteine residues.

1-3. Linker (L)
In formula (I), L is a cleavable linker, a divalent group containing a cleavable moiety.

A cleavable linker represented by L is a divalent group containing a cleavable moiety. A cleavable moiety is a site that can be cleaved by a specific treatment under conditions (mild conditions) that do not cause protein denaturation/degradation (eg, cleavage of amide bonds). Therefore, the cleavable moiety can be said to be a site (bonds other than amide bonds) that can be cleaved by specific cleavage treatment under mild conditions. Examples of such specific treatments include (a) treatment with one or more substances selected from the group consisting of acidic substances, basic substances, reducing agents, oxidizing agents, and enzymes; treatment with a chosen physico-chemical stimulus, or (c) leaving when using a cleavable linker comprising a self-cleavable cleavable moiety. Such cleavable linkers and cleavage conditions thereof are common technical knowledge in the art (e.g., G. Leriche, L. Chisholm, A. Wagner, Bioorganic & Medicinal Chemistry. 20, 571 (2012); Feng P. et al., Journal of American Chemical Society.132, 1500 (2010).; Bessodes M. et al., Journal of Controlled Release, 99, 423 (2004).; 132, 17928 (2010); Thompson, D. H., Journal
of Controlled Release, 91, 187 (2003); G. , Journal of Controlled Release, 95, 291 (2004)). Such cleavable moieties include, for example, disulfide residues, acetal residues, ketal residues, ester residues, carbamoyl residues, alkoxyalkyl residues, imine residues, tertiary alkyloxycarbamate residues (e.g., tert-butyloxycarbamate residue), silane residue, hydrazone-containing residue (e.g. hydrazone residue, acylhydrazone residue, bisarylhydrazone residue), phosphoramidate residue, aconityl residue, trityl residue , an azo residue, a vicinal diol residue, a selenium residue, an aromatic ring-containing residue having an electron withdrawing group, a coumarin-containing residue, a sulfone-containing residue, an unsaturated bond-containing chain residue, and a glycosyl residue. be done.

The aromatic ring group having an electron-withdrawing group preferably has an aromatic ring group selected from the group consisting of aryl, aralkyl, aromatic heterocyclic group, and alkyl having an aromatic heterocyclic group. Alkyl having a cyclic group is more preferred. The electron withdrawing group is preferably attached to the 2-position of the ring. Even more preferably, the aromatic ring-containing residue with an electron withdrawing group is, for example, aralkyl with an electron withdrawing group at the 2-position (eg benzyl). Examples of electron-withdrawing groups include halogen atoms, halogen-substituted alkyls (e.g., trifluoromethyl), boronic acid residues, mesyl, tosyl, triflate, nitro, cyano, phenyl groups, keto groups (e.g., acyl).

Definitions of groups such as alkyl, acyl (i.e. alkylcarbonyl), alkoxy (i.e. alkyloxy), aryl, aralkyl etc. found as prefixes, suffixes etc. terms in connection with the name of the residue as cleavable moiety. , examples, and preferred examples are the same as those described below.

Ester residues include, for example, ordinary ester residues composed of carbon and oxygen atoms [eg, alkyl esters (eg, tertiary alkyloxycarbonyl such as tert-butyloxycarbonyl), aryl esters (eg, phenoxycarbonyl), silester, 2-(diphenylphosphino)benzoate), glycosyl ester residue, orthoester residue, ester residue containing sulfur atom and oxygen atom (e.g., α-thiophenyl ester residue, alkylthioester residue, etc.) thioester residues), ester residues containing phosphorus and oxygen atoms (eg, phosphodiester residues, phosphotriester residues), activated ester residues (eg, N-hydroxysuccinimide residues).

Sulfone-containing residues include, for example, sulfone residues and quinolinylbenzenesulfonate residues.

The silane residue is preferably a silane residue having groups selected from the group consisting of alkyl, aryl, aralkyl and alkoxy. Such silane residues include, for example, dialkyldialkoxysilane residues (eg, dimethyldialkoxysilane, diethyldialkoxysilane) or diaryldialkoxysilane residues (eg, diphenyldialkoxysilane).

Alkoxyalkyl (i.e., alkyloxyalkyl) residues are groups in which alkyloxy and alkyl are combined as described below (definition, examples and preferred examples of alkyloxy and alkyl are the same as those described below), such as methoxy Examples include, but are not limited to, methyl residues, ethoxymethyl residues, methoxyethyl residues, ethoxyethyl residues.

An unsaturated bond-containing chain residue is a residue containing an unsaturated bond portion consisting only of carbon atoms (e.g. vinyl (ethenyl), which is the smallest unit having a double bond between carbon atoms, or a triple bond between carbon atoms acetylenyl (ethynyl), which is the smallest unit, or a residue containing an unsaturated bond moiety (eg, aldehyde, cyano) consisting of a carbon atom and a heteroatom (eg, nitrogen atom, sulfur atom, oxygen atom). Unsaturated bond-containing chain residues include, for example, vinyl ether residues, cyanoethyl residues, ethylene residues, and malondialdehyde residues.

Examples of acidic substances (also called electrophiles) include inorganic acidic substances such as hydrochloric acid, sulfuric acid and nitric acid, and formic acid, acetic acid, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid, 3- Examples include organic acidic substances such as morpholinopropanesulfonic acid, sodium dihydrogen phosphate, citric acid, dodecylsulfuric acid, N-dodecanoylsarcosic acid, and trifluoroacetic acid. Sites cleavable by acidic substances include, for example, alkyloxyarylalkyl residues, tertiary alkyloxycarbamate residues, acetal residues, silane residues, imine residues, vinyl ether residues, and β-thiopropionate residues. groups, trityl residues, hydrazone residues, aconityl residues, orthoester residues, carbamoyl residues, 2-(diphenylphosphino)benzoate residues.

Examples of basic substances (also referred to as nucleophiles) include inorganic basic substances such as sodium hydroxide, potassium hydroxide, sodium acetate, potassium acetate and ammonium acetate, and triethylamine, N,N'-diisopropylamine. organic basic substances such as Sites cleavable by basic substances include, for example, silane residues, cyanoethyl residues, sulfone residues, ethylene residues, glycosyldisuccinate residues, α-thiophenyl ester residues, and unsaturated vinyl sulfide residues. , malondialdehyde residues, acylhydrazone residues, and alkylthioester residues.

Examples of reducing agents include cysteine, dithiothreitol, reduced glutathione, hydroxylamine, and β-mercaptoethanol. Sites that can be cleaved by a reducing agent include, for example, disulfide residues, alkoxyalkyl residues, and azo residues.

Examples of oxidizing agents include sodium periodate and oxidized glutathione. Sites that can be cleaved by an oxidizing agent include, for example, vicinal diol residues and selenium residues.

Enzymes include trypsin, papain, TEV, thrombin, cathepsin B, cathepsin D, cathepsin K, caspase, protease, matrix metalloprotease, lipase, endoglycosidase, PN gauze F, for example. Enzymatically cleavable sites include, for example, ester residues, phosphodiester residues, and glycosyl residues.

Sites cleavable by light include, for example, 2-nitrobenzyl residue, phenacyl ester residue, 8-quinolinebenzenesulfonate residue, coumarin residue, phosphotriester residue, bisarylhydrazone residue, biman A dithiopropionic acid residue can be mentioned.

Autolytic cleavable moieties include, for example, activated ester residues (eg, N-hydroxysuccinimide residues).

〔ここで、結合に直交する波線は、切断部位を示し、
複数のＲ_２ａ、複数のＲ_２ｂ、および複数のＲ_２ｃは、同一または異なって、水素原子、または後述する置換基からなる群より選ばれ、
Ｊは、－ＣＨ_２－、－Ｏ－、または－Ｓ－であり、
ｒは、１～４の任意の整数であり、
○（白丸）はＡ（または後述するＬａ）に対する結合を示し、●（黒丸）はＢ（または後述するＬｂ）に対する結合を示す。
化学構造が、切断部位を中心にして非対称である場合、●がＡ（または後述するＬａ）に対する結合を示し、○がＢ（または後述するＬｂ）に対する結合を示していてもよい。〕 More specifically, the cleavable moiety may correspond to any one chemical structure selected from the group consisting of:

[Where the wavy line perpendicular to the bond indicates the cleavage site,
the plurality of R _2a , the plurality of R _2b , and the plurality of R _2c are the same or different and are selected from the group consisting of hydrogen atoms or substituents described later;
J is —CH ₂ —, —O—, or —S—;
r is any integer from 1 to 4,
O (white circle) indicates binding to A (or La, which will be described later), and ● (black circle) indicates binding to B (or Lb, which will be described later).
If the chemical structure is asymmetrical around the cleavage site, ● may indicate the bond to A (or La, which will be described later), and O may indicate the bond to B (or Lb, which will be described later). ]

J is -CH ₂ -, -O-, or -S-. j is preferably -CH ₂ - or -O-, more preferably -CH ₂ -.

r is any integer from 1 to 4, preferably any integer from 1 to 3, more preferably 1 or 2.

In one embodiment, the cleavable linker is (i) a cleavable linker that is a divalent group comprising a cleavable moiety capable of cleaving to generate a bioorthogonal functional group on the side of the reactive group, or (ii) It may be a cleavable linker that is a divalent group comprising a cleavable moiety that does not have the ability to cleave to generate a bioorthogonal functional group on the side of the reactive group.

Cleavable moieties of (i) include, for example, disulfide residues, ester residues, acetal residues, ketal residues, imine residues, and vicinal diol residues.

〔ここで、結合に直交する波線は、切断部位を示し、
複数のＲ_２ａは、同一または異なって、水素原子、または後述する置換基からなる群より選ばれ、
○（白丸）はＡ（または後述するＬａ）に対する結合を示し、●（黒丸）はＢ（または後述するＬｂ）に対する結合を示す。
化学構造が、切断部位を中心にして非対称である場合、●がＡ（または後述するＬａ）に対する結合を示し、○がＢ（または後述するＬｂ）に対する結合を示していてもよい。〕からなる群より選ばれるいずれか一つの化学構造に対応していてもよい。 More specifically, the cleavable moieties of (i) are, for example:

[Where the wavy line orthogonal to the bond indicates the cleavage site,
a plurality of R _2a are the same or different and are selected from the group consisting of hydrogen atoms or substituents described later;
O (white circle) indicates binding to A (or La, which will be described later), and ● (black circle) indicates binding to B (or Lb, which will be described later).
If the chemical structure is asymmetrical around the cleavage site, ● may indicate the bond to A (or La, which will be described later), and O may indicate the bond to B (or Lb, which will be described later). ] may correspond to any one chemical structure selected from the group consisting of

Cleavable moieties of (ii) include, for example, ester residues, carbamoyl residues, alkoxyalkyl residues, imine residues, tertiary alkyloxycarbamate residues, silane residues, hydrazone-containing residues, phosphoramidate residue, aconityl residue, trityl residue, azo residue, vicinal diol residue, selenium residue, aromatic ring-containing residue with electron-withdrawing group, coumarin-containing residue, sulfone-containing residue, unsaturated bond Including chain residues, glycosyl residues.

〔ここで、結合に直交する波線は、切断部位を示し、
複数のＲ_２ｂ、複数のＲ_２ｃ、Ｊ、ｒは、水素原子、または後述する置換基からなる群より選ばれ、
○（白丸）はＡ（または後述するＬａ）に対する結合を示し、●（黒丸）はＢ（または後述するＬｂ）に対する結合を示す。
化学構造が、切断部位を中心にして非対称である場合、●がＡ（または後述するＬａ）に対する結合を示し、○がＢ（または後述するＬｂ）に対する結合を示していてもよい。〕からなる群より選ばれるいずれか一つの化学構造に対応していてもよい。 More specifically, the cleavable moieties of (ii) are, for example:

[Where the wavy line perpendicular to the bond indicates the cleavage site,
Multiple R _2b , multiple R _2c , J, r are selected from the group consisting of hydrogen atoms or substituents described later,
O (white circle) indicates binding to A (or La, which will be described later), and ● (black circle) indicates binding to B (or Lb, which will be described later).
If the chemical structure is asymmetrical around the cleavage site, ● may indicate the bond to A (or La described later) and O may indicate the bond to B (or Lb described later). ] may correspond to any one chemical structure selected from the group consisting of

In certain embodiments, the cleavable linker (L) may be represented by any one of formulas (L1)-(L3) below:
La-C-Lb (L1)
La-C (L2)
C-Lb (L3)
[In the formula,
La and Lb are each a divalent group,
C is a cleavable moiety. ]

As the divalent group, for example, a divalent hydrocarbon group which may have a substituent, a divalent heterocyclic group which may have a substituent, -C (= O) -, - NR _a — (R _a represents a hydrogen atom or a substituent), —O—, —S—, —C(═S)—, and two or more of these (for example, 2 to 8, preferably 2 to 6, Groups consisting of a combination of 2 to 4) are more preferred.

The divalent hydrocarbon group is a straight-chain, branched-chain or cyclic divalent hydrocarbon group, preferably a straight-chain or branched-chain divalent hydrocarbon group. Divalent hydrocarbon groups include, for example, alkylene, alkenylene, alkynylene, and arylene.

As alkylene, alkylene having 1 to 12 carbon atoms is preferable, alkylene having 1 to 6 carbon atoms is more preferable, and alkylene having 1 to 4 carbon atoms is particularly preferable. The above number of carbon atoms does not include the number of carbon atoms of substituents. Alkylene may be linear, branched, or cyclic, but linear alkylene is preferred. Such alkylenes include, for example, methylene, ethylene, propylene, butylene, pentylene, hexylene.

As alkenylene, alkenylene having 2 to 12 carbon atoms is preferable, alkenylene having 2 to 6 carbon atoms is more preferable, and alkenylene having 2 to 4 carbon atoms is particularly preferable. The above number of carbon atoms does not include the number of carbon atoms of substituents. Alkenylene may be linear, branched or cyclic, but linear alkenylene is preferred. Examples of such alkenylene include ethylenylene, propynylene, butenylene, pentenylene, and hexenylene.

As alkynylene, alkynylene having 2 to 12 carbon atoms is preferable, alkynylene having 2 to 6 carbon atoms is more preferable, and alkynylene having 2 to 4 carbon atoms is particularly preferable. The above number of carbon atoms does not include the number of carbon atoms of substituents. Alkynylene may be linear, branched, or cyclic, but linear alkynylene is preferred. Examples of such alkynylene include ethynylene, propynylene, butynylene, pentynylene, and hexynylene.

As arylene, arylene having 6 to 24 carbon atoms is preferable, arylene having 6 to 18 carbon atoms is more preferable, arylene having 6 to 14 carbon atoms is more preferable, and arylene having 6 to 10 carbon atoms is even more preferable. preferable. The above number of carbon atoms does not include the number of carbon atoms of substituents. Arylene includes, for example, phenylene, naphthylene and anthracenylene.

A divalent heterocyclic group is a divalent aromatic heterocyclic group or a divalent non-aromatic heterocyclic group. The heteroatom constituting the heterocyclic ring preferably contains one or more selected from the group consisting of an oxygen atom, a sulfur atom, a nitrogen atom, a phosphorus atom, a boron atom and a silicon atom, and an oxygen atom, a sulfur atom and a nitrogen atom. It is more preferable to contain one or more selected from the group consisting of

The divalent aromatic heterocyclic group is preferably a divalent aromatic heterocyclic group having 3 to 21 carbon atoms, more preferably a divalent aromatic heterocyclic group having 3 to 15 carbon atoms. A divalent aromatic heterocyclic group having 3 to 9 carbon atoms is more preferable, and a divalent aromatic heterocyclic group having 3 to 6 carbon atoms is even more preferable. The above number of carbon atoms does not include the number of carbon atoms of substituents. More specifically, divalent aromatic heterocyclic groups include, for example, pyrenediyl, pyrroldiyl, furandiyl, thiophenediyl, pyridinediyl, pyridazinediyl, pyrimidinediyl, pyrazinediyl, triazinediyl, pyrrolinediyl, piperidinediyl, triazole Diyl, purinediyl, anthraquinonediyl, carbazolediyl, fluorenediyl, quinolinediyl, and isoquinolinediyl.

The divalent non-aromatic heterocyclic group is preferably a non-aromatic heterocyclic group having 3 to 21 carbon atoms, more preferably a non-aromatic heterocyclic group having 3 to 15 carbon atoms, and 3 to 3 carbon atoms. Non-aromatic heterocyclic groups of 9 are more preferred, and non-aromatic heterocyclic groups of 3 to 6 carbon atoms are even more preferred. The above number of carbon atoms does not include the number of carbon atoms of substituents. More specifically, divalent non-aromatic heterocyclic groups include, for example, pyrroldionediyl, pyrrolinedionediyl, oxranediyl, aziridinediyl, azetidinediyl, oxetanediyl, thietanediyl, pyrrolidinediyl, dihydrofurandiyl, and tetrahydrofurandiyl. , dioxolanediyl, tetrahydrothiophenediyl, imidazolidinediyl, oxazolidinediyl, piperidinediyl, dihydropyrandiyl, tetrahydropyrandiyl, tetrahydrothiopyrandiyl, morpholindiyl, thiomorpholindiyl, piperazinediyl, dihydrooxazinediyl, tetrahydrooxazinediyl, dihydro Pyrimidinediyl, and tetrahydropyrimidinediyl.

The divalent groups represented by La and Lb may have, for example, 1 to 5, preferably 1 to 3, more preferably 1 or 2 substituents. Such substituents are the same as the substituents represented by R _a and R _b above. Such substituents include, for example:
(i) a halogen atom;
(ii) a monovalent hydrocarbon group;
(iii) aralkyl;
(iv) a monovalent heterocyclic group;
(v) R _c -O-, R _c -C(=O)-, R _c -O-C(=O)-, or R _c -C(=O)-O- (R _c is a hydrogen atom or (vi) NR _d R _e -, NR _d R _e -C(=O)-, NR _d R _e -C(=O)-O-, or R _d —C(═O)—NR _e — (R _d and R _e are the same or different and represent a hydrogen atom or a monovalent hydrocarbon group);
(vii) nitro, sulfate, sulfonate, cyano, and carboxyl groups;

Halogen atoms include, for example, fluorine, chlorine, bromine and iodine atoms.

Examples of monovalent hydrocarbon groups include monovalent chain hydrocarbon groups, monovalent alicyclic hydrocarbon groups, and monovalent aromatic hydrocarbon groups.

A monovalent chain hydrocarbon group means a hydrocarbon group composed only of a chain structure, and the main chain does not contain a cyclic structure. However, the chain structure may be linear or branched. Examples of monovalent chain hydrocarbon groups include alkyl, alkenyl, and alkynyl. Alkyl, alkenyl and alkynyl can be straight or branched.

The alkyl is preferably an alkyl having 1 to 12 carbon atoms, more preferably an alkyl having 1 to 6 carbon atoms, and still more preferably an alkyl having 1 to 4 carbon atoms. The above number of carbon atoms does not include the number of carbon atoms of substituents. Examples of alkyl having 1 to 12 carbon atoms include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl and decyl. , dodecyl.

Alkenyl is preferably alkenyl having 2 to 12 carbon atoms, more preferably alkenyl having 2 to 6 carbon atoms, and even more preferably alkenyl having 2 to 4 carbon atoms. The above number of carbon atoms does not include the number of carbon atoms of substituents. Examples of alkenyl having 2 to 12 carbon atoms include vinyl, propenyl and n-butenyl.

Alkynyl is preferably alkynyl having 2 to 12 carbon atoms, more preferably alkynyl having 2 to 6 carbon atoms, and even more preferably alkynyl having 2 to 4 carbon atoms. The above number of carbon atoms does not include the number of carbon atoms of substituents. Examples of alkynyl having 2 to 12 carbon atoms include ethynyl, propynyl and n-butynyl.

Alkyl is preferred as the monovalent chain hydrocarbon group.

A monovalent alicyclic hydrocarbon group means a hydrocarbon group that contains only an alicyclic hydrocarbon as a ring structure and does not contain an aromatic ring. may be However, it does not have to be composed only of alicyclic hydrocarbons, and may partially contain a chain structure. Examples of monovalent alicyclic hydrocarbon groups include cycloalkyl, cycloalkenyl, and cycloalkynyl, which may be either monocyclic or polycyclic.

As cycloalkyl, cycloalkyl having 3 to 12 carbon atoms is preferable, cycloalkyl having 3 to 6 carbon atoms is more preferable, and cycloalkyl having 5 to 6 carbon atoms is even more preferable. The above number of carbon atoms does not include the number of carbon atoms of substituents. Examples of cycloalkyl having 3 to 12 carbon atoms include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

As cycloalkenyl, cycloalkenyl having 3 to 12 carbon atoms is preferable, cycloalkenyl having 3 to 6 carbon atoms is more preferable, and cycloalkenyl having 5 to 6 carbon atoms is even more preferable. The above number of carbon atoms does not include the number of carbon atoms of substituents. Cycloalkenyl having 3 to 12 carbon atoms includes, for example, cyclopropenyl, cyclobutenyl, cyclopentenyl and cyclohexenyl.

As cycloalkynyl, cycloalkynyl having 3 to 12 carbon atoms is preferable, cycloalkynyl having 3 to 6 carbon atoms is more preferable, and cycloalkynyl having 5 to 6 carbon atoms is even more preferable. The above number of carbon atoms does not include the number of carbon atoms of substituents. Cycloalkynyl having 3 to 12 carbon atoms includes, for example, cyclopropynyl, cyclobutynyl, cyclopentynyl and cyclohexynyl.

Cycloalkyl is preferred as the monovalent alicyclic hydrocarbon group.

A monovalent aromatic hydrocarbon group means a hydrocarbon group containing an aromatic ring structure. However, it does not have to be composed only of aromatic rings, and may contain a chain structure or alicyclic hydrocarbon in part, and the aromatic ring may be monocyclic or polycyclic. good. The monovalent aromatic hydrocarbon group is preferably an aryl having 6 to 12 carbon atoms, more preferably an aryl having 6 to 10 carbon atoms, and still more preferably an aryl having 6 carbon atoms. The above number of carbon atoms does not include the number of carbon atoms of substituents. Examples of aryl having 6 to 12 carbon atoms include phenyl and naphthyl.

Phenyl is preferred as the monovalent aromatic hydrocarbon group.

Among these, the monovalent hydrocarbon group is preferably alkyl, cycloalkyl or aryl, more preferably alkyl.

Aralkyl refers to arylalkyl. Definitions, examples and preferred examples of aryl and alkyl in arylalkyl are as described above. As aralkyl, aralkyl having 3 to 15 carbon atoms is preferable. Such aralkyls include, for example, benzoyl, phenethyl, naphthylmethyl, naphthylethyl.

A monovalent heterocyclic group refers to a group obtained by removing one hydrogen atom from a heterocyclic ring of a heterocyclic compound. A monovalent heterocyclic group is a monovalent aromatic heterocyclic group or a monovalent non-aromatic heterocyclic group. The heteroatom constituting the heterocyclic group preferably contains one or more selected from the group consisting of an oxygen atom, a sulfur atom, a nitrogen atom, a phosphorus atom, a boron atom and a silicon atom, and an oxygen atom, a sulfur atom and a nitrogen atom. More preferably, it contains one or more selected from the group consisting of atoms.

The monovalent aromatic heterocyclic group is preferably an aromatic heterocyclic group having 3 to 15 carbon atoms, more preferably an aromatic heterocyclic group having 3 to 9 carbon atoms, and an aromatic heterocyclic group having 3 to 6 carbon atoms. Group heterocyclic groups are more preferred. The above number of carbon atoms does not include the number of carbon atoms of substituents. Monovalent aromatic heterocyclic groups include, for example, pyrenyl, pyrrolyl, furanyl, thiophenyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrrolinyl, piperidinyl, triazonyl, purinyl, carbazonyl, fluorenyl, quinolinyl, and isoquinolinyl. .

The monovalent non-aromatic heterocyclic group is preferably a non-aromatic heterocyclic group having 3 to 15 carbon atoms, more preferably a non-aromatic heterocyclic group having 3 to 9 carbon atoms, and 3 to 3 carbon atoms. 6 non-aromatic heterocyclic groups are more preferred. The above number of carbon atoms does not include the number of carbon atoms of substituents. Examples of monovalent non-aromatic heterocyclic groups include oxiranyl, aziridinyl, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, dihydrofuranyl, tetrahydrofuranyl, dioxolanyl, tetrahydrothiophenyl, imidazolidinyl, oxazolidinyl, piperidinyl, dihydropyranyl, tetrahydro Pyranyl, tetrahydrothiopyranyl, morpholinyl, thiomorpholinyl, piperazinyl, dihydrooxazinyl, tetrahydrooxazinyl, dihydropyrimidinyl, and tetrahydropyrimidinyl.

Among these, the monovalent heterocyclic group is preferably a 5- or 6-membered heterocyclic group.

Preferably, the substituents may be:
(i') a halogen atom;
(ii') alkyl having 1 to 12 carbon atoms, phenyl, or naphthyl;
(iii') aralkyl having 3 to 15 carbon atoms;
(iv') a 5- or 6-membered heterocyclic ring;
(v') R _c -O-, R _c -C(=O)-, R _c -O-C(=O)-, or R _c -C(=O)-O- (R _c is hydrogen or (vi') NR _d R _e -, NR d R e -C(=O)-, NR _{d R e -C} (=O)- O—, or R _d —C(═O)—NR _e — (R _d and R _e are the same or different and represent a hydrogen atom or alkyl having 1 to 12 carbon atoms.);
(vii') the same groups as listed in (vii) above.

More preferably, the substituents may be:
(i'') a halogen atom;
(ii'') alkyl having 1 to 12 carbon atoms;
(iii'') R _c -O-, R _c -C(=O)-, R _c -O-C(=O)-, or R _c -C(=O)-O- (R _c is represents a hydrogen atom or alkyl having 1 to 12 carbon atoms.); or (iv'') NR _d R _e —, NR _d R _e —C(=O)—, NR _d R _e —C(=O )—O—, or R _d —C(═O)—NR _e — (R _d and R _e are the same or different and represent a hydrogen atom or alkyl having 1 to 12 carbon atoms.);
(v'') The same groups as listed in (vii) above.

Even more preferably, the substituents may be:
(i''') a halogen atom;
(ii''') alkyl having 1 to 6 carbon atoms;
(iii''') R _c -O-, R _c -C(=O)-, R _c -O-C(=O)-, or R _c -C(=O)-O- (R _c is , a hydrogen atom, _or alkyl having _{1 to} 6 carbon atoms _{. ) ;} =O)-O-, or R _d -C(=O)-NR _e - (R _d and R _e are the same or different and represent a hydrogen atom or alkyl having 1 to 6 carbon atoms);
(v''') the same groups as those enumerated in (vii) above.

Particularly preferably, the substituents may be:
(i'''') a halogen atom;
(ii'''') alkyl having 1 to 4 carbon atoms;
(iii'''') R _c -O-, R _c -C(=O)-, R _c -O-C(=O)-, or R _c -C(=O)-O-(R _c represents a hydrogen atom or alkyl having 1 to 4 carbon atoms.); or (iv'''') NR _d R _e -, NR _d R _e -C(=O)-, NR _d R _e - C(=O)-O-, or R _d -C(=O)-NR _e - (R _d and R _e are the same or different and represent a hydrogen atom or alkyl having 1 to 4 carbon atoms. );
(v'''') the same groups as listed in (vii) above.

〔式中、
ｐおよびｐ’は、同一または異なって、０～１０の任意の整数であり、
ｑおよびｑ’は、同一または異なって、０～１０の任意の整数であり、
ＸおよびＸ’は、同一または異なって、炭素原子、窒素原子、または単結合（ここで、Ｘが窒素原子である場合、Ｒ_１ｂは存在せず、Ｘ’が窒素原子である場合、Ｒ_１ｂ’は存在しない。Ｘが単結合である場合、Ｒ_１ａおよびＲ_１ｂは存在せず、Ｘ’が単結合である場合、Ｒ_１ａ’およびＲ_１ｂ’は存在しない）であり、
Ｒ_１ａ、Ｒ_１ｂ、Ｒ_１ａ’およびＲ_１ｂ’は、同一または異なって、水素原子、または上述する置換基からなる群より選ばれる。〕で表されてもよい。 In certain embodiments, La and Lb are (La') and (Lb'), respectively:

[In the formula,
p and p' are the same or different and any integer from 0 to 10;
q and q' are the same or different and any integer from 0 to 10;
X and X' are the same or different and are a carbon atom, a nitrogen atom, or a single bond (wherein when X is a nitrogen atom, R _1b is absent, and when X' is a nitrogen atom, R _{1b '} is absent.If X is a single bond, R _1a and R _1b are absent, and if X' is a single bond, R _1a' and R _1b' are absent);
R _1a , R _1b , R _1a′ and R _1b′ are the same or different and are selected from the group consisting of a hydrogen atom or the substituents described above. ] may be represented.

p and p' are the same or different and are any integer from 0 to 10, preferably an integer from 0 to 8, more preferably an integer from 0 to 6, still more preferably an integer from 0 to 4 It is an integer, particularly preferably 0, 1 or 2. Preferably p and p' are the same.

q and q' are the same or different and are any integer from 0 to 10, preferably an integer from 0 to 8, more preferably an integer from 0 to 6, still more preferably an integer from 0 to 4 It is an integer, particularly preferably 0, 1 or 2. Preferably q and q' are the same.

X and X' are the same or different and are a carbon atom, a nitrogen atom, or a single bond, preferably a carbon atom or a single bond. Preferably X and X' are the same.

R _1a , R _1b , R _1a' and R _1b' are the same or different and are selected from the group consisting of hydrogen atoms or substituents described later. Definitions, examples and preferred examples of substituents are as described above. Preferably, R _1a , R _1b , R _1a′ and R _1b′ are hydrogen atoms.

1-4. (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group (B) that does not contain a bioorthogonal functional group
In formula (I), B is (a) a divalent group that contains a bioorthogonal functional group or (b) a divalent group that does not contain a bioorthogonal functional group.

Bioorthogonal functional groups do not react with biological constituents (e.g., amino acids, nucleic acids, lipids, sugars, phosphoric acids), or react slowly with biological constituents, but react with components other than biological constituents. A group that selectively reacts with Bioorthogonal functional groups are well known in the art (e.g., Sharpless KB et al., Angew. Chem. Int. Ed. 40, 2004 (2015); Bertozzi C. R. et al., Science 291, 2357 (2001); Bertozzi CR et al., Nature Chemical Biology 1, 13 (2005)).

If the target of the affinity agent is a soluble protein, the bioorthogonal functional group is a bioorthogonal functional group to the protein. A bioorthogonal functional group for a protein is a group that reacts with a predetermined functional group without reacting with the side chains of the 20 naturally occurring amino acid residues that constitute the protein. The 20 natural amino acids that make up proteins are alanine (A), asparagine (N), cysteine (C), glutamine (Q), glycine (G), isoleucine (I), leucine (L), methionine (M ), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), valine (V), aspartic acid (D), glutamic acid (E), arginine ( R), histidine (H), and lysine (L). Of these 20 naturally occurring amino acids, glycine without a side chain (i.e., a hydrogen atom) and from the group consisting of a sulfur atom, a nitrogen atom, and an oxygen atom, in which the side chain is a hydrocarbon group (i.e., Alanine, isoleucine, leucine, phenylalanine, and valine, which do not contain the selected heteroatom in their side chains, are inert to normal reactions. Bioorthogonal functional groups for proteins are thus asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, in addition to those amino acid side chains with side chains that are inert to normal reactions. , aspartic acid, glutamic acid, arginine, histidine, and lysine.

Such bioorthogonal functional groups that cannot react with proteins include, for example, azide residues, aldehyde residues, thiol residues, alkene residues (in other words, the smallest unit having a double bond between carbon atoms It suffices if it has a certain vinylene (ethynylene) moiety, hereinafter the same), alkyne residue (in other words, it suffices if it has an ethynylene moiety, which is the minimum unit having a triple bond between carbon atoms, hereinafter the same) , halogen residue, tetrazine residue, nitrone residue, hydroxylamine residue, nitrile residue, hydrazine residue, ketone residue, boronic acid residue, cyanobenzothiazole residue, allyl residue, phosphine residue, maleimide residue, disulfide residue, thioester group, α-halocarbonyl residue (e.g., a carbonyl residue having a fluorine atom, chlorine atom, bromine atom or iodine atom at the α-position; the same shall apply hereinafter), isonitrile residue, sydone residue , selenium residues. Proteins include proteins that can contain free thiols (cysteine) (eg, proteins other than antibodies) and proteins that cannot contain free thiols (eg, antibodies). Thiols function as bioorthogonal functional groups in proteins that cannot contain free thiols. Therefore, when the soluble protein that is the target of the affinity substance is a protein (eg, antibody) that cannot contain free thiols, the bioorthogonal functional group preferably contains thiols. Also, when the soluble protein is a protein that can contain free thiols (eg, proteins other than antibodies), it is preferred that the bioorthogonal functional groups do not contain thiols. One or more (e.g., two, three, four) bioorthogonal functional groups may be contained in the divalent group, but preferably one bioorthogonal functional group is It may be contained in a divalent group.

In one embodiment, the divalent groups comprising bioorthogonal functional groups are azide residues, aldehyde groups, thiol residues, alkyne residues, alkene residues, tetrazine residues, nitrone residues, hydroxylamine residues, nitrile residue, hydrazine residue, ketone residue, boronic acid residue, cyanobenzothiazole residue, allyl residue, phosphine residue, maleimide residue, disulfide residue, thioester group, α-halocarbonyl residue, isonitrile It may be a divalent group containing, in its main chain, a bioorthogonal functional group selected from the group consisting of residues, sydnon residues, and selenium residues.

In another embodiment, the divalent group comprising a bioorthogonal functional group is an azide residue, an aldehyde residue, a thiol residue, an alkyne residue, an alkene residue, a halogen residue, a tetrazine residue, a nitrone residue. , hydroxylamine residue, nitrile residue, hydrazine residue, ketone residue, boronic acid residue, cyanobenzothiazole residue, allyl residue, phosphine residue, maleimide residue, disulfide residue, α-halocarbonyl residue It may be a divalent group containing a bioorthogonal functional group selected from the group consisting of groups, isonitrile residues, sydnon residues, and selenium residues in the side chain.

〔式中、
Ｒ_１ｆ、単一もしくは複数のＲ_１ｇおよび単一もしくは複数のＲ_１ｈは、同一もしくは異なって、前記（ｉ）～（ｖｉｉ）からなる群より選ばれる原子もしくは基、または電子吸引基であり、
・は、結合手である。） More specifically, the bioorthogonal functional group may correspond to any one chemical structure selected from the group consisting of:

[In the formula,
R _1f , single or multiple R _1g and single or multiple R _1h are the same or different and are atoms or groups selected from the group consisting of (i) to (vii), or electron-withdrawing groups;
・ is a bond. )

Examples of the electron-withdrawing group include those described above, and halogen atoms, boronic acid residues, mesyl, tosyl, and triflate are preferred.

In one embodiment, B may be (a) a divalent group comprising a bioorthogonal functional group. The number of bioorthogonal functional groups contained in the divalent group may be single or multiple, eg 1-5, preferably 1-3, more preferably 1 or 2, even more preferably 1. When the number of bioorthogonal functional groups contained in the divalent group is plural, the plural bioorthogonal functional groups may be of the same type or different types. Preferably, they are of the same type.

In certain embodiments, B may be (a1) a divalent group comprising a bioorthogonal functional group in its backbone. Bivalent groups containing bioorthogonal functional groups in the main chain include azide residues, aldehyde groups, thiol residues, alkyne residues, alkene residues, tetrazine residues, nitrone residues, hydroxylamine residues, and nitrile residues. group, hydrazine residue, ketone residue, boronic acid residue, cyanobenzothiazole residue, allyl residue, phosphine residue, maleimide residue, disulfide residue, thioester group, α-halocarbonyl residue, isonitrile residue , a sydnon residue, and a selenium residue, a bioorthogonal functional group itself being a divalent group, or at either end or both ends of such a bivalent bioorthogonal functional group It is a group to which the divalent groups described above are linked. The definition, examples and preferred examples of the divalent group to be linked are the same as for the divalent group described above.

In another particular embodiment, B may be (a2) a divalent group containing a bioorthogonal functional group in its side chain. Bivalent groups containing bioorthogonal functional groups in side chains include azide residues, aldehyde residues, thiol residues, alkyne residues, alkene residues, halogen residues, tetrazine residues, nitrone residues, hydroxylamine residue, nitrile residue, hydrazine residue, ketone residue, boronic acid residue, cyanobenzothiazole residue, allyl residue, phosphine residue, maleimide residue, disulfide residue, α-halocarbonyl residue, isonitrile It is a divalent group substituted with a bioorthogonal functional group selected from the group consisting of a residue, a sydnon residue and a selenium residue, or a group containing the same. The definition, examples and preferred examples of the divalent group to be substituted are the same as for the divalent group described above.

In another embodiment, B may be (b) a divalent group that does not contain a bioorthogonal functional group. Such divalent groups include optionally substituted alkylene, optionally substituted cycloalkylene, optionally substituted aryl, optionally substituted divalent heterocyclic group, —NR _a - (R _a represents a hydrogen atom or a substituent), -O-, and a group consisting of a combination of two or more thereof (for example, 2 to 8, preferably 2 to 6, more preferably 2 to 4). may The optionally substituted substituents and the substituents of R _a are substituents other than bioorthogonal functional groups. Such substituents include, for example, alkyl, cycloalkyl, aralkyl, monovalent heterocyclic groups, hydroxyl, amino, alkyloxy (alkoxy), cycloalkyloxy, aralkyloxy. The number of such substituents is, for example, 1 to 5, preferably 1 to 3, more preferably 1 or 2, still more preferably 1.

Definitions, examples and preferred examples of alkyl, cycloalkyl, aralkyl and monovalent heterocyclic groups for substituents other than bioorthogonal functional groups are as described above.

Regarding substituents other than bioorthogonal functional groups, definitions, examples and preferred examples of alkyl in alkyloxy (alkoxy), cycloalkyl in cycloalkyloxy, and aralkyl in aralkyloxy are as described above. More specifically, alkyloxy includes, for example, methyloxy, ethyloxy, propyloxy (eg, n
-propyloxy, iso-propyloxy), butyloxy (e.g. n-butyloxy, iso-butyloxy, sec-butyloxy, tert-butyloxy), pentyloxy (e.g. n-pentyloxy), hexyloxy (e.g. n-hexyl) oxy). Cycloalkyloxy includes, for example, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy and cyclohexyloxy. Aralkyloxy includes, for example, benzoyloxy, phenethyloxy, naphthylmethyloxy, naphthylethyloxy.

A divalent group that does not contain a bioorthogonal functional group may also be a group that is highly inert to reactions. Such divalent groups may therefore be groups composed only of carbon and hydrogen atoms. Such divalent groups are alkylene, cycloalkylene, or aryl, and combinations of two or more thereof (eg, 2 or 3). When the divalent group that does not contain a bioorthogonal functional group is a group that is highly inert to the reaction, such a divalent group can be alkylene as the highly inert substituent to the reaction. , cycloalkylene, and aryl. The number of substituents highly inert to the reaction is, for example, 1-5, preferably 1-3, more preferably 1 or 2.

〔式中、
Ｙは、－ＮＨ－、－Ｏ－、－ＣＨ_２－、または下記式（Ｂ－２）：

（式中、
ＶおよびＶ’は、同一または異なって、－ＮＨ－、－Ｏ－、－ＣＨ_２－、または単結合であり、
Ｖ１は、（ａ）生体直交性官能基を含む２価の基、または（ｂ）生体直交性官能基を含まない２価の基であり、
ｓは、０～１０の任意の整数であり、
式（Ｂ－２）における○および●は、それぞれ、式（Ｂ－１）における○および●と同じ配向である。）であり、
Ｚは、酸素原子、硫黄原子、または水素原子（Ｚが水素原子である場合、－Ｃ（＝Ｚ）－は、－ＣＨ_２－を示す。）であり、
式（Ｂ－１）における○（白丸）は、Ｌ側の部分に対する結合を示し、●（黒丸）はＲ側の部分に対する結合を示す。〕で表されてもよい。 In certain embodiments, B is the following formula (B-1):

[In the formula,
Y is -NH-, -O-, -CH ₂ -, or the following formula (B-2):

(In the formula,
V and V′ are the same or different and are —NH—, —O—, —CH ₂ —, or a single bond;
V is (a) a divalent group containing a bioorthogonal functional group or (b) a divalent group without a bioorthogonal functional group;
s is any integer from 0 to 10,
○ and ● in formula (B-2) have the same orientations as ○ and ● in formula (B-1), respectively. ) and
Z is an oxygen atom, a sulfur atom, or a hydrogen atom (when Z is a hydrogen atom, -C(=Z)- represents -CH ₂ -);
○ (white circle) in formula (B-1) indicates a bond to the L-side portion, and ● (black circle) indicates a bond to the R-side portion. ] may be represented.

Y is —NH—, —O—, —CH ₂ —, or a group represented by formula (B-2) above. From the viewpoint of structural simplification, Y may be -NH-, -O-, or -CH ₂ -. Alternatively, from the viewpoint of designing a structure based on a carbon atom, Y may be —CH ₂ — or a group represented by the above formula (B-2).

Z is an oxygen atom, a sulfur atom, or a hydrogen atom, preferably an oxygen atom or a sulfur atom.

V and V' are -NH-, -O-, -CH ₂ -, or a single bond, preferably -CH ₂ -, or a single bond.

V1 is (a) a divalent group that includes a bioorthogonal functional group or (b) a divalent group that does not include a bioorthogonal functional group. Such divalent groups are the same as those described above.

Preferably, the divalent group for V1 is an optionally substituted divalent hydrocarbon group or an optionally substituted divalent heterocyclic group. The definition, examples and preferred examples of the divalent hydrocarbon group are the same as those described above, but in the case of V1, alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene and arylene are preferred. For example, alkenylene, alkynylene, cycloalkenylene, cycloalkynylene are preferred if they are not substituted with moieties containing bioorthogonal functional groups. On the other hand, when substituted with moieties containing bioorthogonal functional groups, alkylene, cycloalkylene and arylene are preferred. Examples and preferred examples of these groups are as described above. The definition, examples and preferred examples of the divalent heterocyclic group are the same as those described above, but in the case of V1, a 5- or 6-membered heterocyclic group is preferred. Examples and preferred examples of the 5- or 6-membered heterocyclic group are the same as those mentioned above. Definitions, examples and preferred examples of substituents are as described above. V1 may have (a) bioorthogonal functional groups, for example 1 to 5, preferably 1 to 3, more preferably 1 or 2, even more preferably 1. When the number of bioorthogonal functional groups contained in the divalent group is plural, the plural bioorthogonal functional groups may be of the same type or different types. From the viewpoint of adopting a simple structure and improving reactivity, it is preferable that they are of the same type. From the viewpoint of ensuring differentiated reactions, etc., it is preferable that the cells are of different types. V1 may also have 1 to 5, preferably 1 to 3, more preferably 1 or 2 (b) substituents.

s is any integer from 0 to 10, preferably an integer from 0 to 8, more preferably an integer from 0 to 6, even more preferably an integer from 0 to 4, particularly preferably 0 , 1 or 2.

〔式中、
ＧおよびＧ’は、同一または異なって、－ＮＨ－、－Ｏ－、－ＣＨ_２－、単結合、または下記式（Ｂ－４）：

（式中、
ＷおよびＷ’は、同一または異なって、－ＮＨ－、－Ｏ－、－ＣＨ_２－、または単結合であり、
Ｗ１は、（ａ）生体直交性官能基を含む２価の基、または（ｂ）生体直交性官能基を含まない２価の基であり、
ｔは、０～１０の任意の整数である。
式（Ｂ－４）における○（白丸）は、式（Ｂ－３）における結合手（・）の方向に対する結合を示し、●（黒丸）はｂ側の方向に対する結合を示す。）で表される基であり、
Ｈは、－ＣＨ_２－、－Ｃ＝Ｏ－、－Ｃ＝Ｓ－、－ＮＨ－、または単結合であり、
Ｉは、２価の炭化水素基、２価の複素環、または単結合であり、
ｂは、下記：

（式中、
Ｒ_１ｆ、単一もしくは複数のＲ_１ｇおよび単一もしくは複数のＲ_１ｈは、同一もしくは異なって、前記（ｉ）～（ｖｉｉ）からなる群より選ばれる原子もしくは基、または電子吸引基であり、
・は、結合手である。）で表されるいずれか一つの基である。〕で表される基を側鎖として有する２価の基であってもよい。このような２価の基は、２価の炭化水素基、または２価の複素環基であり、好ましくは、置換されていてもよい２価の炭化水素基であり、より好ましくは、アルキレン、アルケニレン、アルキニレン、シクロアルキレン、シクロアルケニレン、シクロアルキニレン、またはアリーレンであり、さらより好ましくは、アルキレン、シクロアルキレン、またはアリーレンであり、特に好ましくはアルキレンである。これらの基の例および好ましい例は、上述したとおりである。これらの基は、上記側鎖以外の置換基で置換されていてもよい。このような置換基の数は、１～５個、好ましくは１～３個、より好ましくは１もしくは２個である。置換基の例および好ましい例は、上述したとおりである。 In a more specific embodiment, V1 has the following formula (B-3):

[In the formula,
G and G' are the same or different, -NH-, -O-, -CH ₂ -, a single bond, or the following formula (B-4):

(In the formula,
W and W′ are the same or different and are —NH—, —O—, —CH ₂ —, or a single bond;
W1 is (a) a divalent group containing a bioorthogonal functional group or (b) a divalent group without a bioorthogonal functional group;
t is any integer from 0-10.
○ (white circle) in formula (B-4) indicates a bond in the direction of the bond (·) in formula (B-3), and ● (black circle) indicates a bond in the b-side direction. ) is a group represented by
H is —CH ₂ —, —C═O—, —C═S—, —NH—, or a single bond;
I is a divalent hydrocarbon group, a divalent heterocyclic ring, or a single bond;
b is:

(In the formula,
R _1f , single or multiple R _1g and single or multiple R _1h are the same or different and are atoms or groups selected from the group consisting of (i) to (vii), or electron-withdrawing groups;
・ is a bond. ) is any one group represented by ] as a side chain may be a divalent group. Such a divalent group is a divalent hydrocarbon group or a divalent heterocyclic group, preferably an optionally substituted divalent hydrocarbon group, more preferably alkylene, It is alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene or arylene, more preferably alkylene, cycloalkylene or arylene, particularly preferably alkylene. Examples and preferred examples of these groups are as described above. These groups may be substituted with substituents other than the above side chains. The number of such substituents is 1-5, preferably 1-3, more preferably 1 or 2. Examples and preferred examples of substituents are as described above.

G and G' are the same or different and are -NH-, -O-, -CH ₂ -, a single bond, or a group represented by formula (B-4) above. From the viewpoint of structural simplification, G and G' may be -NH-, -O-, -CH ₂ -, or a single bond. Alternatively, from the viewpoint of designing a structure based on a carbon atom, G and G' may be —CH ₂ —, a single bond, or a group represented by formula (B-4) above.

H is -CH ₂ -, -C=O-, -C=S-, -NH-, or a single bond. Preferably H is -CH ₂ - or a single bond.

I is a divalent hydrocarbon group, a divalent heterocyclic ring, or a single bond. A divalent hydrocarbon group and a divalent heterocyclic ring may or may not be substituted with a substituent. Definitions, examples and preferred examples of the divalent hydrocarbon group, divalent heterocyclic ring, and substituent are the same as those described above for V1.

W and W' are the same or different and are -NH-, -O-, -CH ₂ - or a single bond, preferably -CH ₂ - or a single bond.

W1 is (a) a divalent group that includes a bioorthogonal functional group or (b) a divalent group that does not include a bioorthogonal functional group. Such divalent groups are the same as those described above.

t is any integer from 0 to 10, preferably an integer from 0 to 8, more preferably an integer from 0 to 6, even more preferably an integer from 0 to 4, particularly preferably 0 , 1 or 2.

1-5. Reactive group (R)
In formula (I), R is a reactive group for soluble proteins. Such reactive groups are common technical knowledge in the technical field.

Reactive groups are the same or different groups as the bioorthogonal functional groups.

For example, if B is (a) a divalent group comprising a bioorthogonal functional group, the reactive group may be a group dissimilar to the bioorthogonal functional group. This is because if the reactive group is the same kind of group as the bioorthogonal functional group, the reaction specificity of the reactive group with respect to the soluble protein cannot be ensured. In addition, the bioorthogonal functional group is originally a group that cannot react with side chains of 20 natural amino acid residues that constitute soluble proteins.

More specifically, among the 20 natural amino acids that constitute proteins, glycine without a side chain, and alanine, isoleucine, leucine, phenylalanine, and valine with a hydrocarbon group in the side chain are Inactive to normal reactions. Thus, reactive groups for proteins are any one or two of the 14 amino acids consisting of asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine. It is a group capable of reacting with the above (eg, 2, 3, 4) side chains. Depending on conditions such as the amino acid composition of the protein, one or more (e.g., two, three, four) reactive groups may be contained in the compound represented by formula (I). However, preferably one reactive group may be included in the compound of formula (I).

Preferably, the reactive group is a group capable of reacting with the side chain of any one of the 14 amino acids that constitute proteins.

More preferably, the reactive group may be a reactive group specific for the side chain of any one of lysine, tyrosine, tryptophan, or cysteine amino acids.

Even more preferably, the reactive group may be a reactive group specific for the side chain of any one of lysine, tyrosine or tryptophan amino acids.

Also, when the protein is human IgG, such as human IgG1, the reactive group is preferably a reactive group specific for the side chains of lysine or tyrosine.

A reactive group specific for the side chain of a lysine residue is a group capable of specifically reacting with an amino group (NH ₂ ) present in the side chain of a lysine residue, such as an activated ester residue (e.g., N-hydroxysuccinimide residue), vinyl sulfone residue, sulfonyl chloride residue, isocyanate residue, isothiocyanate residue, aldehyde residue, 1,4,7,10-tetraazacyclododecane-1,4 , 7,10-tetraacetic acid residue, 2-imino-2-methoxyethyl residue, and diazonium terephthalic acid residue.

The linking moiety generated by the reaction between the above-mentioned reactive group specific to the side chain of the lysine residue and the amino group (NH ₂ ) present in the side chain of the lysine residue includes, for example, Amide residues, urea residues, pyridine residues, carbamate residues, sulfonamide residues can be mentioned.

〔ここで、
Ｒ_５ａおよびＲ_５cは、水素原子、または上述した置換基であり、
Ｒ_５ｂは、電子吸引基であり、
ｊは、１～５の任意の整数であり、
ｋは、１～４の任意の整数である。〕 More specifically, the reactive group specific for the side chain of a lysine residue may correspond to any one chemical structure selected from the group consisting of:

〔here,
R _5a and R _5c are hydrogen atoms or substituents as described above,
R _5b is an electron withdrawing group,
j is any integer from 1 to 5;
k is any integer from 1 to 4. ]

_R5a and _R5c are hydrogen atoms or substituents as described above. Definitions, examples and preferred examples of substituents are the same as those described above.

_R5b is an electron withdrawing group. Examples of such electron-withdrawing groups include those described above, and halogen atoms, boronic acid residues, mesyl, tosyl, and triflate are preferred.

j is any integer from 1 to 5, preferably an integer from 1 to 3, more preferably 1 or 2;

k is any integer from 1 to 4, preferably an integer from 1 to 3, more preferably 1 or 2.

〔ここで、●（黒丸）は、Ｔ側の部分に対する結合を示し、○（白丸）は、Ｂ側の部分に対する結合を示す。結合に直交する直線は、反応により生成する結合を示す。〕 The linking moiety generated by the reaction between the above chemical structure, which is a reactive group specific to the side chain of the lysine residue, and the amino group (NH ₂ ) present in the side chain of the lysine residue, It may correspond to any one chemical structure selected from the group consisting of the following.

[Here, ● (black circle) indicates a bond to the portion on the T side, and O (white circle) indicates a bond to the portion on the B side. A straight line perpendicular to the bond indicates the bond produced by the reaction. ]

A reactive group specific for the side chain of a tyrosine residue is a group capable of specifically reacting with an atom ortho to a phenolic hydroxyl group (OH) present in the side chain of a tyrosine residue, e.g. , diazonium residue, diazodicarboxylate residue, 2,3-dihydro-1H-pyrazin-6-one residue.

〔ここで、Ｒ_４ａは、水素原子、または上述した置換基であり、○は、Ｂに対する結合を示す。〕 More specifically, the reactive group specific for the side chain of a tyrosine residue may correspond to any one chemical structure selected from the group consisting of:

[Here, R _4a is a hydrogen atom or a substituent as described above, and ○ indicates a bond to B. ]

R _4a is a hydrogen atom or a substituent as described above. Definitions, examples and preferred examples of substituents are the same as those described above.

〔ここで、Ｒ_４ａは、水素原子、または上述した置換基であり、●は、Ｔに対する結合を示し、○は、Ｂに対する結合を示す。〕 By reaction between the above chemical structure, which is a reactive group specific for the side chain of tyrosine residues, and the atom ortho to the phenolic hydroxyl group (OH) present in the side chains of tyrosine residues. The resulting linking moiety may correspond to any one chemical structure selected from the group consisting of:

[Here, R _4a is a hydrogen atom or a substituent as described above, ● indicates a bond to T, and ○ indicates a bond to B. ]

R _4a is a hydrogen atom or a substituent as described above. Definitions, examples and preferred examples of substituents are the same as those described above.

The reactive group specific to the side chain of tryptophan residue is a group capable of specifically reacting with the 3-position ring-constituting atom of the indole group present in the side chain of tryptophan residue, for example, 9- and azabicyclo[3.3.1]nonan-3-one-N-oxyl residues.

〔ここで、○は、Ｂに対する結合を示す。〕 More specifically, the reactive group specific for the side chain of a tryptophan residue may correspond to any one chemical structure selected from the group consisting of:

[Here, O indicates a bond to B. ]

〔ここで、●は、Ｔに対する結合を示し、○は、Ｂに対する結合を示す。〕 A linkage formed by a reaction between the above chemical structure, which is a reactive group specific to the side chain of a tryptophan residue, and the ring atom at the 3-position of the indole group present in the side chain of the tryptophan residue. A moiety may correspond to any one chemical structure selected from the group consisting of:

[Here, ● indicates a bond to T and O indicates a bond to B. ]

Particularly preferably, the reactive group may be a reactive group specific for the side chain of lysine.

1-6. Partial structure "LB"
1-6-1. Length of main chain in partial structure “LB” connecting A and R In formula (I), main chain connecting A (affinity substance) and R (reactive group) (L The length of the linear portion) is determined by the type of soluble protein and affinity substance, and the target region of the affinity substance in the soluble protein to which R should react and bind, as described above (e.g., It can be appropriately designed according to various factors such as the relationship with the position and number of specific amino acid residues in the specific position). In the compound represented by formula (I), an affinity substance is associated with a soluble protein, and then a reactive group covalently bonded to the affinity substance via LB is present in the vicinity of the target site. can be covalently attached to soluble proteins by reacting with groups in the side chains of specific amino acid residues (eg, amino groups in the side chains of lysine residues). At this time, the specific amino acid in the region near the specific amino acid residue to which R should react and bind, the region near the target site, and the region between the specific amino acid residue and the target site If the residue is not otherwise present, the reactive group can be regioselectively attached to that particular amino acid residue without strict control of backbone length. Of course, by controlling the length of the backbone, the reactive group can be positioned relative to the particular amino acid residue, even if the particular amino acid residue is otherwise present in such regions. Can be selectively bound.

The length of the backbone linking A and R depends on factors such as the type of soluble protein and its affinity substance, and the position and number of specific amino acid residues in the target site in the soluble protein. Although it can vary, it may be about 5 Å or greater, preferably about 7.5 Å or greater, and more preferably about 10.5 Å or greater. Such backbone lengths may also be, for example, about 30 Å or less, preferably about 23 Å or less, more preferably about 16.5 Å or less. More specifically, the length of such backbone may be, for example, about 5.0-30 Å, preferably about 7.5-23 Å, more preferably about 10.5-16.5 Å.

By the way, it is common general knowledge in this technical field that the relationship of the length (distance) between atoms is as shown in the table below. Therefore, a person skilled in the art can appropriately design a main chain having the number of atoms corresponding to the length (Å) of the main chain as described above, referring to the interatomic lengths shown in the table below. .

More specifically, the length of the main chain connecting A and R can also be defined as the number of atoms constituting the main chain (excluding hydrogen atoms and substituents). The number of atoms constituting the main chain may be, for example, 4 (about 5.0 Å) or more, preferably 6 (about 7.5 Å), more preferably 8 (about 10.5 Å) or more. The number of atoms in the backbone may also be, for example, 20 (about 30 Å) or less, preferably 16 (about 23 Å) or less, more preferably 12 (about 16.5 Å) or less. More specifically, the number of atoms in the main chain may be, for example, 4-20, preferably 6-16, more preferably 8-12.

When the main chain is a structure that does not contain a ring structure, the number of atoms in the main chain can be determined by counting the number of atoms in the chain structure.

・は、結合手である。
（ａ）の場合、最短経路は太字経路であるため、主鎖の原子数として数えられる２価の環構造中の原子数は、２である。
（ｂ）の場合、最短経路は太字経路であるため、主鎖の原子数として数えられる２価の環構造中の原子数は、３である。
（ｃ）の場合、いずれの経路も最短経路（等距離）であるため、主鎖の原子数として数えられる２価の環構造中の原子数は、４である。
（ｄ）の場合、縮合部位の経路が最短経路であるため、主鎖の原子数として数えられる２価の環構造中の原子数は、４である。 On the other hand, when the main chain has a structure containing a ring structure, the number of atoms in the main chain does not necessarily correspond to the length described above, and the length that can be defined by the number of atoms in the main chain is shorter than the length described above. Tend. Even in such a case, the number of atoms in the main chain can be conveniently counted from the viewpoint of defining the length of the main chain. Specifically, the number of atoms in the main chain in such cases is the number of atoms in the chain structure that does not contain a divalent ring structure in the main chain, plus the number of atoms that connect two bonds in the ring structure. It can be determined by counting the number of atoms in the shortest path (see, eg, bold-faced paths in (a)-(d) below).

・ is a bond.
In the case of (a), since the shortest route is the bold-faced route, the number of atoms in the divalent ring structure counted as the number of atoms in the main chain is 2.
In the case of (b), since the shortest route is the bold-faced route, the number of atoms in the divalent ring structure counted as the number of atoms in the main chain is 3.
In the case of (c), all paths are the shortest paths (equidistant), so the number of atoms in the divalent ring structure counted as the number of atoms in the main chain is 4.
In the case of (d), the number of atoms in the divalent ring structure counted as the number of atoms in the main chain is 4, since the path of the condensation site is the shortest path.

Preferably, the linking portion between A and R represented by LB (excluding the side chain) may be a chain structure that does not contain a divalent ring structure. In this case, L and B can be appropriately designed so that the linking chain portion between A and R represented by LB does not contain a divalent ring group.

1-6-2. Specific Structure of Partial Structure “LB” In the above formula (I), L and B are structures that can be associated with each other. Therefore, in the above formula (I), L and B can be defined as a partial structure represented by "LB".

In one embodiment, the cleavable linker is (i) a cleavable linker that is a divalent group comprising a cleavable moiety capable of cleaving to generate a bioorthogonal functional group on the side of the reactive group, or (ii) It may be a cleavable linker that is a divalent group comprising a cleavable moiety that does not have the ability to cleave to generate a bioorthogonal functional group on the side of the reactive group.

In certain embodiments, when L is the cleavable linker of (i) above, B is (a) a divalent group comprising a bioorthogonal functional group, or (b) free of a bioorthogonal functional group. It is a divalent group.

Preferably, when L is the cleavable linker of (i) above, B is (a) a divalent group comprising a bioorthogonal functional group. In this case, the bioorthogonal functional group produced in (i) may be the same or different from the bioorthogonal functional group in (a). From the viewpoint of adopting a simpler structure and/or improving reactivity with a single functional substance, the bioorthogonal functional group generated in (i) is different from the bioorthogonal functional group in (a). They may be of the same species. On the other hand, from the viewpoint of ensuring differentiated reactivity with two or more functional substances and not using some of the bioorthogonal functional groups in the reaction, the bioorthogonal functional groups generated in (i) may be heterologous to the bioorthogonal functional groups in (a).

Alternatively, when L is the cleavable linker of (i) above, B may be (b) a divalent group that does not contain a bioorthogonal functional group. In this case, the compound represented by formula (I) or a salt thereof has a simpler structure, and is easy to synthesize.

In another specific embodiment, when L is the cleavable linker of (ii) above, B is (a
) is a divalent group containing a bioorthogonal functional group.

In certain embodiments, the substructure represented by LB preferably does not include peptide moieties. In this case, the advantage is that the soluble protein (eg, antibody drug conjugate) having the functional substance of the present invention obtained using the compound of the present invention may not contain a peptide moiety that may have immunogenicity as a linker. be.

In a specific embodiment, the partial structure represented by "LB" is an atom (e.g., the main chain If the number of atoms constituting the main chain is an odd number), or the binding site that exists at the central position of the main chain (e.g., if the number of atoms constituting the main chain is an even number), the symmetrical structure [e.g. , Z), trans (ie, E)]. For example, a centrally located binding site can be designed as a cleavable moiety in a cleavable linker as described above. When the partial structure represented by "LB" has a symmetrical structure, the partial structure represented by "LB" can be easily synthesized. For example, by reacting the same divalent groups having at one end a functional group capable of reacting to form a cleavable moiety (e.g., a chain divalent group having an SH group at one end), A symmetrical structure (chained divalent group—S—S—chained divalent group) containing a cleavable moiety at the central position of the main chain can be realized.

Therefore, the partial structure represented by "LB" is the partial structure represented by "B2-L'-B1" (that is, L is a divalent group represented by B2-L', B is B1.). In this case, the compound represented by the above formula (I) can be defined as a compound represented by the following formula (I').
AB2-L'-B1-R (I')
[In the formula,
A and R are the same as in formula (I) above L′ is a cleavable linker that is a divalent group containing a cleavable moiety;
B1 and B2 are the same or different and are (a) a divalent group containing a bioorthogonal functional group or (b) a divalent group containing no bioorthogonal functional group;
B1 and B2 may have a symmetrical structure about L'. ]

B1 and B2 are the same or different and have the same definitions, examples and preferred examples of B.

In certain embodiments, L' may be represented by any one of formulas (L1') to (L3') below:
La'-C'-Lb'(L1')
La'-C'(L2')
C'-Lb'(L3')
[In the formula,
La' and Lb' are each a divalent group,
C' is a cleavable moiety. ]

The divalent groups represented by La' and Lb' are the same as the definition, examples and preferred examples of the divalent groups represented by La' and Lb', respectively.

The cleavable moiety represented by C' is the same as the definition, examples and preferred examples of the cleavable moiety represented by C.

〔式中、
Ｃ、ｐ、ｐ’、ｑ、ｑ’、Ｘ、Ｘ’、Ｒ_１ａ、Ｒ_１ａ’、Ｒ_１ｂ、Ｒ_１ｂ’、Ｙ、Ｙ’、ＺおよびＺ’の定義、例および好ましい例は、上述したものと同じであり、
○（白丸）は、Ａに対する結合を示し、●（黒丸）は、Ｒに対する結合を示す。〕 In another specific embodiment, the structural unit represented by LB may be represented by the following formula (LB').

[In the formula,
Definitions, examples and preferred examples of C, p, p', q, q', X, X', R _1a , R _1a' , R _1b , R _1b' , Y, Y', Z and Z' are given above. is the same as
O (white circle) indicates a bond to A and ● (filled circle) indicates a bond to R. ]

〔式中、
Ａ、Ｒ、Ｃ、ｐ、ｐ’、ｑ、ｑ’、Ｘ、Ｘ’、Ｒ_１ａ、Ｒ_１ｂ、Ｒ_１ａ’、Ｒ_１ｂ’、Ｙ、Ｙ’、ＺおよびＺ’の定義、例および好ましい例は、上述したものと同じである。〕 Therefore, the above formula (I) can be defined as the following formula (I'').

[In the formula,
Definitions, examples and preferences of A, R, C, p, p', q, q', X, X', R _1a , R _1b , R _1a' , R _1b' , Y, Y', Z and Z' Examples are the same as described above. ]

In the above formulas (LB') and (I''), the length from C (carbon atom) in C=Z to C (carbon atom) in C=Z' is the length of the main chain connecting A and R is the same as In these formulas, the length from C in C=Z to C in C=Z' also refers to the connecting chain of the substructure connecting C in C=Z to C in C=Z' (hydrogen atoms and substituents ) can also be defined as the number of atoms constituting The number of such atoms is the same as the number of atoms constituting the main chain connecting A and R. The connecting chain of the partial structure connecting C in C=Z to C in C=Z' (excluding hydrogen atoms and substituents) may or may not contain a ring structure, but may contain a ring structure. preferably not included. The linking chain (excluding hydrogen atoms and substituents) may preferably be free of peptide moieties.

1-8. Production Method A compound or a salt thereof containing an affinity substance for a soluble protein, a cleavable moiety and a reactive group can be prepared as appropriate. A compound comprising an affinity agent for a soluble protein, a cleavable moiety and a reactive group is represented by formula (I), preferably formula (I'), more preferably formula (I'').

As the affinity substance, one having any functional group can be appropriately selected. Therefore, by utilizing a reactive group capable of reacting with the functional group, the affinity substance can be converted into a structural unit represented by LBR or a structure represented by RLB A structural unit represented by ALBR can be prepared by reacting units (wherein the two reactive groups are the same or different). For example, such reactions can be carried out in a suitable reaction system, such as an organic solvent system or an aqueous system, at a suitable temperature (eg, about 15-200° C.). The reaction system may contain a suitable catalyst. The reaction time is, for example, 1 minute to 20 hours, preferably 10 minutes to 15 hours, more preferably 20 minutes to 10 hours, still more preferably 30 minutes to 8 hours.

In the reaction system, the molar ratio (Y/X) of the structural unit represented by LBR or the structural unit (Y) represented by RLBR to the affinity substance (X) is , the types of the structural unit and the affinity substance, the number of sites in the affinity substance to be modified by the structural unit, and the like, but are not particularly limited, but are preferably 0.1 to 50, for example. is 0.5 to 40, more preferably 1 to 35, even more preferably 2 to 25, and particularly preferably 3 to 15.

Confirmation of production of a soluble protein or salt thereof comprising an affinity substance for a soluble protein and a cleavable moiety can be performed, for example, by electrophoresis, chromatography (e.g., Gel filtration chromatography, ion exchange chromatography, reverse phase column chromatography, HPLC) or mass spectrometry, preferably mass spectrometry. Affinity agents for soluble proteins and soluble proteins or salts thereof containing cleavable moieties can be suitably purified by any method such as chromatography (eg, chromatography as described above, and affinity chromatography).

1-9. Other inventions described later [e.g., formulas (II) to (v) and subordinate concept formulas thereof, and partial structural formulas (e.g., (L1) to (L3), (La'), (Lb'), (B- 1) to (B-4))], arbitrary symbols (eg, A, L, B, R), terms represented by the symbols, and their details (eg, definitions, examples and preferred examples) are in common with the invention of the compound represented by formula (I) or a salt thereof. In addition, specific moieties (e.g., cleavable moieties, moieties having the ability to generate bioorthogonal functional groups on the reactive group side by cleavage), specific groups (e.g., bioorthogonal functional groups, divalent groups, alkyl groups, substituents, electron withdrawing groups), and specific numerical values, as well as optional technical elements such as salts (eg, definitions, examples and preferred examples) are also described above. can be made in common. Therefore, these matters can be used as appropriate in the invention described later without particular reference. Similarly, the technical elements of specific inventions described in the later-described inventions can be appropriately incorporated as the technical elements of the present invention and other inventions.

2. Soluble protein containing affinity substance for soluble protein and cleavable moiety or salt thereof 2-1. SUMMARY The present invention provides a soluble protein or salt thereof comprising an affinity agent for a soluble protein and a cleavable moiety of formula (II).
A-LB-R'-T (II)
[In the formula,
A is an affinity substance for soluble proteins;
L is a cleavable linker, which is a divalent group containing a cleavable moiety;
B is (a) a divalent group that includes a bioorthogonal functional group or (b) a divalent group that does not include a bioorthogonal functional group;
R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein. ]

2-2. Moiety (R') generated by reaction between soluble protein and reactive group
Definitions, examples and preferred examples of the soluble protein and the reactive group in the moiety generated by the reaction between the soluble protein and the reactive group are given above. The portion generated by the reaction between the soluble protein and the reactive group is common technical knowledge in the technical field, and can be appropriately determined according to the type of soluble protein and reactive group.

Preferably, the moieties generated by the reaction between the soluble protein and the reactive group are 14 amino acids that can be contained in the soluble protein (asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine), the moiety generated by the reaction between the side chain of any one amino acid and a reactive group thereto.

More preferably, the moiety generated by the reaction between the soluble protein and the reactive group is an amino acid side chain of any one of lysine, tyrosine, tryptophan, or cysteine and a reactive group specific thereto. It may be a portion generated by a reaction between.

Even more preferably, the moiety generated by the reaction between the soluble protein and the reactive group is an amino acid side chain of any one of lysine, tyrosine, or tryptophan and a reactive group specific therefor. It may be a portion generated by a reaction between Examples of the portion generated by the reaction between the side chain of any one of lysine, tyrosine, or tryptophan and a reactive group specific thereto include the above-mentioned "1-5. Reactive group (R)” linking moieties and/or chemical structures.

Even more preferably, the moiety generated by reaction between a soluble protein and a reactive group is a moiety generated by reaction between a lysine or tyrosine side chain and a reactive group specific therefor. (particularly if the soluble protein is human IgG, such as human IgG1). Examples of the moiety generated by the reaction between the side chain of lysine or tyrosine and the reactive group specific thereto include the linking moiety and the and/or chemical structures.

Particularly preferably, the moiety generated by reaction between a soluble protein and a reactive group may be a moiety generated by reaction between a side chain of lysine and a reactive group specific therefor. .

2-3. Partial structure "LB"
The details of the partial structure “LB” are as described above in “1-6. Partial structure “LB””.

In certain embodiments, the substructure represented by "LB" is a substructure represented by "B2-L'-B1" (i.e., L is a divalent and B is B1). In this case, the affinity substance for the soluble protein represented by the above formula (II) and the soluble protein containing the cleavable moiety or a salt thereof can be represented by the following formula (II').
AB2-L'-B1-R'-T (II')
[In the formula,
A, R' and T are the same as in formula (II) above L' is a cleavable linker that is a divalent group containing a cleavable moiety;
B1 and B2 are the same or different and are (a) a divalent group containing a bioorthogonal functional group or (b) a divalent group without a bioorthogonal functional group;
B1 and B2 may have a symmetrical structure about L'. ]

In formula (II') above, L' may be represented by any one of formulas (L1') to (L3') described above.

〔式中、
Ｃ、ｐ、ｐ’、ｑ、ｑ’、Ｘ、Ｘ’、Ｒ_１ａ、Ｒ_１ａ’、Ｒ_１ｂ、Ｒ_１ｂ’、Ｙ、Ｙ’、ＺおよびＺ’の定義、例および好ましい例は、上述したものと同じであり、
○（白丸）は、Ａに対する結合を示し、●（黒丸）は、Ｒ’に対する結合を示す。〕 In another specific embodiment, the structural unit represented by LB may be represented by the following formula (LB').

[In the formula,
Definitions, examples and preferred examples of C, p, p', q, q', X, X', R _1a , R _1a' , R _1b , R _1b' , Y, Y', Z and Z' are given above. is the same as
O (open circle) indicates binding to A and ● (filled circle) indicates binding to R'. ]

〔式中、
Ａ、Ｒ’、Ｔ、Ｃ、ｐ、ｐ’、ｑ、ｑ’、Ｘ、Ｘ’、Ｒ_１ａ、Ｒ_１ｂ、Ｒ_１ａ’、Ｒ_１ｂ’、Ｙ、Ｙ’、ＺおよびＺ’の定義、例および好ましい例は、上述したものと同じである。〕 Therefore, the above formula (II) can be defined as the following formula (II'').

[In the formula,
definitions of A, R′, T, C, p, p′, q, q′, X, X′, R _1a , R _1b , R _1a′ , R _1b′ , Y, Y′, Z and Z′; Examples and preferred examples are the same as described above. ]

In formulas (LB') and (II'') above, the length from C (carbon atom) in C=Z to C (carbon atom) in C=Z' connects A and R as described above. It is the same as the main chain length. In these formulas, the length from C in C=Z to C in C=Z' also refers to the connecting chain of the substructure connecting C in C=Z to C in C=Z' (hydrogen atoms and substituents ) can also be defined as the number of atoms constituting The number of such atoms is the same as the number of atoms constituting the main chain connecting A and R. The connecting chain of the partial structure connecting C in C=Z to C in C=Z' (excluding hydrogen atoms and substituents) may or may not contain a ring structure, but may contain a ring structure. preferably not included. The linking chain (excluding hydrogen atoms and substituents) may preferably be free of peptide moieties.

2-4. Binding sites of partial structures other than soluble proteins possessed by soluble proteins (regioselectivity)
Partial structures other than soluble proteins (eg, ALBR') can be regioselectively bound to target regions as described above in soluble proteins (T).

As used herein, "regioselectivity" or "regioselectivity" refers to specific amino acid residues in a soluble protein, even though the specific amino acid residues are not unevenly distributed in a specific region in the soluble protein. It means that a predetermined structural unit capable of binding to is unevenly distributed in a specific region in a soluble protein. Thus, expressions related to regioselectivity such as "regioselectively having", "regioselectively binding", "regioselectively binding", etc., refer to target regions comprising one or more specific amino acid residues. is significantly higher than the retention or binding rate of a given structural unit in a non-target region containing multiple amino acid residues that are homologous to the specific amino acid residue in the target region It means that it is high at a certain level. Such regioselective binding or retention is directed to specific amino acid residues in a target region in a soluble protein rather than randomly reacting a given structural unit to specific amino acid residues in a soluble protein. This can be achieved by the present invention in which a predetermined structural unit can be reacted preferentially.

Specifically, T contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and the specific amino acid residue in a non-target region other than the target region When it contains 5 or more amino acid residues, the partial structure other than the soluble protein can bind to one or more specific amino acid residues contained in the target region with a position selectivity of 30% or more. . Definitions, examples and preferred examples of target regions and regioselectivity are provided above.

2-5. Number of partial structures other than soluble protein possessed by soluble protein The number of partial structures (eg, ALBR') possessed by the soluble protein (T) other than the soluble protein can vary. For example, if T is a multimeric protein comprising a plurality of monomeric proteins, T can have substructures other than T in the corresponding plurality of target regions in the plurality of monomeric proteins. . As a result, T can have multiple partial structures other than T. Therefore, the structure represented by formula (II), (II'), or (II'') indicates that T may have one or more partial structures other than T. The number of partial structures other than T that T has can be adjusted by appropriately setting conditions such as the type of soluble protein and the reaction ratio between the soluble protein and structural units introduced therein. Such number may vary depending on the type of soluble protein, but may be, for example, 1 to 8, preferably 1 to 4, more preferably 1 or 2.

In certain embodiments, if the soluble protein is a multimeric protein composed of a plurality of monomeric proteins, the number of partial structures other than the soluble protein possessed by the soluble protein may be more than one. good. According to the present invention, a plurality of partial structures other than soluble proteins can be introduced into the same target region of a plurality of monomeric proteins.

In preferred embodiments, the soluble protein may be an antibody comprising multiple heavy chains. Definitions, examples and preferred examples of antibodies are given above. The number of heavy chains varies depending on the type of antibody. For example, IgG, IgE, and IgD can have two heavy chains. IgA can have two or four heavy chains. IgM can have 8 heavy chains. The number of non-antibody partial structures possessed by an antibody (soluble protein) can be considered synonymous with DAR. In the present invention, the number of partial structures other than antibodies possessed by antibodies is 1 or 2 (preferably 2) for IgG, IgE, and IgD, and 1 to 4 (preferably 4), and for IgM may be 1 to 8 (preferably 8).

2-6. Methods of Making The present invention provides methods of making a soluble protein or salt thereof comprising an affinity agent for a soluble protein and a cleavable moiety, comprising:
(A1) reacting a compound or a salt thereof containing a soluble protein affinity substance, a cleavable moiety and a reactive group with a soluble protein to produce a soluble protein affinity substance and a soluble protein comprising a cleavable moiety or a salt thereof; to generate.

A compound comprising an affinity agent for a soluble protein, a cleavable moiety and a reactive group is represented by formula (I), preferably formula (I'), more preferably formula (I''). The affinity agent for soluble proteins and the soluble protein comprising the cleavable moiety is represented by formula (II), preferably formula (II'), more preferably formula (II'').

Compounds or salts thereof containing affinity substances for soluble proteins, cleavable moieties and reactive groups can react with soluble proteins because they have reactive groups. Such a reaction can be appropriately carried out under conditions (mild conditions) that do not cause protein denaturation/decomposition (eg, cleavage of amide bonds). For example, such reactions can be carried out at room temperature (eg, about 15-30° C.) in a suitable reaction system, eg, a buffer. The pH of the buffer is, for example, 5-9, preferably 5.5-8.5, more preferably 6.0-8.0. The buffer may contain a suitable catalyst. The reaction time is, for example, 1 minute to 20 hours, preferably 10 minutes to 15 hours, more preferably 20 minutes to 10 hours, still more preferably 30 minutes to 8 hours. For details of such reactions, see, for example, G.I. J. L. Bernardes et al. , Chem. Rev. , 115, 2174 (2015); J. L. Bernardes et al. , Chem. Asian. J. , 4, 630 (2009); G. Davies et al. , Nat. Commun. , 5, 4740 (2014); Wagner et al. , Bioconjugate. Chem. , 25, 825 (2014).

In the reaction system, the molar ratio (Y/X) of the soluble protein-affinity substance, the cleavable moiety and the compound containing the reactive group or its salt (Y) to the soluble protein (X) is , types of compounds and soluble proteins (e.g., antibodies) containing cleavable moieties and reactive groups, affinity substances for soluble proteins, sites in soluble proteins to be modified by compounds containing cleavable moieties and reactive groups. Although it is not particularly limited because it varies depending on the number (eg, DAR) etc., it is, for example, 0.1 to 100, preferably 0.5 to 80, more preferably 1 to 70, and even more preferably is 2-50, particularly preferably 3-30.

Confirmation of production of a soluble protein or salt thereof comprising an affinity substance for a soluble protein and a cleavable moiety can be performed, for example, by electrophoresis, chromatography (e.g., Gel filtration chromatography, ion exchange chromatography, reverse phase column chromatography, HPLC) or mass spectrometry, preferably mass spectrometry. Confirmation of regioselectivity can be performed, for example, by peptide mapping. Peptide mapping can be performed, for example, by protease (eg, trypsin, chymotrypsin) treatment and mass spectrometry. As the protease, an endoprotease is preferred. Such endoproteases include, for example, trypsin, chymotrypsin, Glu-C, Lys-N, Lys-C, Asp-N. Confirmation of the number of partial structures other than the soluble protein that the soluble protein has can be performed, for example, by electrophoresis, chromatography, or mass spectrometry, preferably by mass spectrometry. Affinity agents for soluble proteins and soluble proteins or salts thereof containing cleavable moieties can be suitably purified by any method such as chromatography (eg, chromatography as described above, and affinity chromatography).

3. Complex or salt thereof having affinity substance for soluble protein, cleavable moiety, functional substance and soluble protein 3-1. Overview The present invention provides a conjugate or salt thereof having an affinity agent for a soluble protein, a cleavable moiety, a functional agent and a soluble protein represented by Formula (III).
ALB'(-F)-R'-T (III)
[In the formula,
A is an affinity substance for soluble proteins;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B' is a divalent group that includes a moiety generated by a reaction between a functional agent and a bioorthogonal functional group;
F is a functional substance,
R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein. ], a complex having an affinity substance for a soluble protein, a cleavable moiety, a functional substance and a soluble protein, or a salt thereof.

In formula (III) or other formulas, R' is covalently attached to B' instead of F.

3-2. Functional substance (F)
The functional substance is not particularly limited as long as it imparts an arbitrary function to the soluble protein. Examples thereof include drugs, labeling substances, and stabilizers, preferably drugs or labeling substances. A functional substance may also be a single functional substance, or a substance in which two or more functional substances are linked.

The drug may be a drug for any disease. Such diseases include, for example, cancer (e.g., lung cancer, stomach cancer, colon cancer, pancreatic cancer, kidney cancer, liver cancer, thyroid cancer, prostate cancer, bladder cancer, ovarian cancer, uterine cancer, bone cancer, skin cancer, brain tumor, melanoma), autoimmune diseases/inflammatory diseases (e.g., allergic diseases, rheumatoid arthritis, systemic lupus erythematosus), cranial nerve diseases (e.g., cerebral infarction, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis), Infectious diseases (e.g., bacterial infections, viral infections), hereditary/rare diseases (e.g., hereditary spherocytosis, non-dystrophic myotonia), eye diseases (e.g., age-related macular degeneration, diabetic retinopathy, retinitis pigmentosa), bone/orthopedic diseases (e.g., osteoarthritis), blood diseases (e.g., leukemia, purpura), other diseases (e.g., diabetes, metabolic disorders such as hyperlipidemia) , liver disease, renal disease, pulmonary disease, circulatory system disease, digestive system disease). When the soluble protein is an antibody against a target protein of a given disease, the drug is a drug related to the given disease (e.g., a drug for treating the given disease, an antibody against the target protein of the given disease drugs that alleviate associated side effects).

More specifically, the drug is an anticancer agent. Anti-cancer agents include, for example, chemotherapeutic agents, toxins, radioactive isotopes or substances containing the same. Chemotherapeutic agents include, for example, DNA damaging agents, antimetabolites, enzyme inhibitors, DNA intercalating agents, DNA cleaving agents, topoisomerase inhibitors, DNA binding inhibitors, tubulin binding inhibitors, cytotoxic nucleosides, A platinum compound is mentioned. Toxins include, for example, bacterial toxins (eg, diphtheria toxin), plant toxins (eg, ricin). Radioisotopes include, for example, a hydrogen atom radioisotope (e.g., ³ H), a carbon atom radioisotope (e.g., ¹⁴ C), a phosphorus atom radioisotope (e.g., ³² P), and a sulfur atom radioisotope (e.g., 32 P). Radioisotopes (e.g. 35 ^S ), yttrium radioisotopes (e.g. ⁹⁰ Y), technetium radioisotopes (e.g. ^99m Tc), indium radioisotopes (e.g. ¹¹¹ In), iodine atom radioactivity Isotopes (e.g., ¹²³ I, ¹²⁵ I, ¹²⁹ I, ¹³¹ I), radioisotopes of samarium (e.g., ¹⁵³ Sm), radioisotopes of rhenium (e.g., ¹⁸⁶ Re), radioisotopes of astatine (e.g., ²¹¹ At), and radioactive isotopes of bismuth (eg, ²¹² Bi).

Examples of labeling substances include enzymes (e.g., peroxidase, alkaline phosphatase, luciferase, β-galactosidase), affinity substances (e.g., streptavidin, biotin, digoxigenin, aptamers), fluorescent substances (e.g., fluorescein, fluorescein isothiocyanate, rhodamine , green fluorescent protein, red fluorescent protein), luminescent substances (e.g., luciferin, aequorin, acridinium ester, tris(2,2'-bipyridyl)ruthenium, luminol), radioisotopes (e.g., those described above) or A substance containing

The functional substance is also a high-molecular compound, a medium-molecular compound, or a low-molecular compound, preferably a low-molecular compound. A low-molecular-weight compound means a compound having a molecular weight of 1,500 or less. Small compounds are natural or synthetic compounds. The molecular weight of the low molecular compound may be 1200 or less, 1000 or less, 900 or less, 800 or less, 700 or less, 600 or less, 500 or less, 400 or less, or 300 or less. The molecular weight of the small molecule compound may also be 30 or greater, 40 or greater, or 50 or greater. A low-molecular-weight compound may be a drug or labeling substance as described above. Examples of low-molecular-weight compounds include amino acids, oligopeptides, vitamins, nucleosides, nucleotides, oligonucleotides, monosaccharides, oligosaccharides, lipids, fatty acids, and salts thereof.

3-3. A divalent group (B') comprising a moiety generated by a reaction between a functional substance and a bioorthogonal functional group

B' is the same as (a) the divalent group containing the bioorthogonal functional group in B described above, except that the bioorthogonal functional group (a) reacts with the functional substance. Therefore, definitions, examples and preferred examples of divalent groups and orthogonal functional groups in B' are the same as in B.

A functional substance has various functional groups according to its structure. When the functional substance has a functional group that readily reacts with the bioorthogonal functional group, the functional group of the functional substance and the bioorthogonal functional group can be appropriately reacted. Functional groups that are reactive with bioorthogonal functional groups may also vary depending on the specific type of bioorthogonal functional group. A person skilled in the art can appropriately select an appropriate functional group as a functional group that readily reacts with the bioorthogonal functional group. Functional groups that readily react with bioorthogonal functional groups include, for example, maleimide and disulfide when the bioorthogonal functional group is thiol, and azide when the bioorthogonal functional group is alkyne. Examples include, but are not limited to, hydrazine when the functional group is an aldehyde or ketone.

On the other hand, when the functional substance does not have a functional group that readily reacts with the bioorthogonal functional group, the functional substance derivatized to have a desired functional group can be used. For example, when the functional substance is a soluble protein, it can be used that is derivatized to have a functional group not naturally possessed by the soluble protein. In this case derivatization of the soluble protein may be performed by the method of the invention. In this case, a soluble protein regioselectively derivatized to have the desired bioorthogonal functional groups can be used as the functional substance.

Derivatization is common knowledge in the art (eg, WO2004/010957, US2006/0074008, US2005/0238649). For example, derivatization may be performed using a cross-linking agent as described above. Alternatively, derivatization may be performed with specific linkers bearing desired functional groups. For example, such a linker may be capable of separating the functional substance and the soluble protein in an appropriate environment (eg, intracellular or extracellular) by cleaving the linker. Such linkers include, for example, peptidyl linkers ( Dubowchik et al., Pharm.Therapeutics 83:67-123 (1999)), linkers that can be cleaved at local acidic sites present in vivo (e.g., US Patent No. 5 , 622,929, 5,122,368; 5,824,805). Linkers may be self-immolative (eg, WO02/083180, WO04/043493, WO05/112919). In the present invention,
A derivatized functional substance can also be simply referred to as a "functional substance".

Definitions, examples and preferred examples of the functional substance and the bioorthogonal functional group in the divalent group containing the moiety generated by the reaction between the functional substance and the bioorthogonal functional group are as described above. The moieties generated by the reaction between the functional substance and the bioorthogonal functional group are common general knowledge in the art, and the functional substance (when the functional substance is derivatized, its derivatized portion) and It can be appropriately determined according to the type of bioorthogonal functional group.

The divalent group including the portion generated by the reaction between the functional substance and the bioorthogonal functional group is not particularly limited, but examples include disulfide residues, acetal residues, ketal residues, esters residues, carbamoyl residues, alkoxyalkyl residues, imine residues, tertiary alkyloxycarbamate residues, silane residues, hydrazone-containing residues, phosphoramidate residues, aconityl residues, trityl residues, azo residues group, a vicinal diol residue, a selenium residue, an aromatic ring-containing residue having an electron-withdrawing group, a coumarin-containing residue, a sulfone-containing residue, an unsaturated bond-containing chain residue, and a glycosyl residue. It may be a divalent group containing a residue that can be

〔ここで、結合に直交する波線は、反応により生成する結合を示し、
複数のＲ_２ａ、複数のＲ_２ｂ、および複数のＲ_２ｃは、同一または異なって、上述した水素原子または置換基であり、
Ｊは、－ＣＨ_２－、－Ｏ－、または－Ｓ－であり、
ｒは、１～４の任意の整数であり、
○（白丸）はＦ側の部分に対する結合を示し、●（黒丸）はＢ’側の部分に対する結合
を示す。
化学構造が、切断性部分を中心にして非対称である場合、●がＢ’側の部分に対する結合を示し、○がＦ側の部分に対する結合を示していてもよい。〕からなる群より選ばれるいずれか一つの化学構造に対応する残基を含む２価の基であってもよい。 Divalent groups, including moieties generated by reaction between a functional agent and a bioorthogonal functional group, are also not particularly limited and include, but are not limited to:

[Here, the wavy line orthogonal to the bond indicates the bond generated by the reaction,
the plurality of R _2a , the plurality of R _2b , and the plurality of R _2c are the same or different and are hydrogen atoms or substituents as described above;
J is —CH ₂ —, —O—, or —S—;
r is any integer from 1 to 4,
O (white circle) indicates a bond to the F-side moiety, and ● (black circle) indicates a bond to the B'-side moiety.
If the chemical structure is asymmetric about the cleavable moiety, the circle may indicate the bond to the B' side moiety and the circle to the F side moiety. ] may be a divalent group containing a residue corresponding to any one chemical structure selected from the group consisting of

3-4. Partial structure "LB'(-F)"
The details of the partial structure "LB'(-F)" are as described in "1-6. Partial structure "LB"" above, except that B is changed to B'(-F). That's right.

In one embodiment, the cleavable linker is (i) a cleavable linker that is a divalent group comprising a cleavable moiety capable of cleaving to generate a bioorthogonal functional group on the side of the reactive group, or (ii) It may be a cleavable linker that is a divalent group comprising a cleavable moiety that does not have the ability to cleave to generate a bioorthogonal functional group on the side of the reactive group.

In certain embodiments, the cleavable linker may be the cleavable linker of (ii) above. This is because B' is already bound to the functional substance (F), so there is no need to generate a bioorthogonal functional group on the side of the reactive group.

In another specific embodiment, the substructure represented by LB'(-F) is the substructure represented by B2'(-F2)-L'-B1'(-F1) (i.e., L is B2'(-F2)-L' and B' is B1'). In this case, the complex or salt thereof having the affinity substance for the soluble protein, the cleaving moiety, the functional substance and the soluble protein represented by the above formula (III) is represented by the following formula (III'). can be done. A-B2'(-F2)-L'-B1'(-F1)-R'-T (III')
[In the formula,
A, R' and T are the same as in formula (III) above;
L' is a cleavable linker that is a divalent group containing a cleavable moiety;
B1' and B2' are the same or different and are divalent groups comprising moieties generated by reaction between a functional agent and a bioorthogonal functional group;
F1 and F2 are the same or different and are functional substances;
B1' (-F1) and B2' (-F2) may have a symmetrical structure about L'. ].

B1' and B2' are the same or different and have the same definitions, examples and preferred examples of B'.

In formula (III') above, L' may be represented by any one of formulas (L1') to (L3') described above.

〔式中、
Ｃ、Ｆ１、Ｆ２、ｐ、ｐ’、ｑ、ｑ’、Ｘ、Ｘ’、Ｒ_１ａ、Ｒ_１ａ’、Ｒ_１ｂ、Ｒ_１ｂ’、ＺおよびＺ’の定義、例および好ましい例は、上述したものと同じであり、
ＹおよびＹ’は、同一または異なって、前記式（Ｂ－１）のＹから水素原子を一つ除いた残基であり、
○（白丸）は、Ａに対する結合を示し、●（黒丸）は、Ｒ’に対する結合を示す。〕 In another specific embodiment, the structural unit represented by LB'(-F) may be represented by the following formula (LB'(F)').

[In the formula,
Definitions, examples and preferred examples of C, F1, F2, p, p', q, q', X, X', R _1a , R _1a' , R _1b , R _1b' , Z and Z' are given above. is the same as
Y and Y' are the same or different and are a residue obtained by removing one hydrogen atom from Y in the formula (B-1),
O (open circle) indicates binding to A and ● (filled circle) indicates binding to R'. ]

More specifically, in Y and Y', the residue obtained by removing one hydrogen atom from Y in the formula (B-1) is -N(-)-, -CH(-)-, or the above formula It is a group obtained by removing one hydrogen atom from (B-2). From the viewpoint of structural simplification, Y may be -N(-)- or -CH(-)-. Alternatively, from the viewpoint of designing a structure based on carbon atoms, Y may be -CH(-)- or a group obtained by removing one hydrogen atom from the above formula (B-2).

（式中、
ＶおよびＶ’は、同一または異なって、－ＮＨ－、－Ｏ－、－ＣＨ_２－、または単結合であり、
Ｖ１は、（ａ）生体直交性官能基を含む２価の基、または（ｂ）生体直交性官能基を含まない２価の基であり、
ｓは、０～１０の任意の整数であり、
式（Ｂ－２’）における○および●は、それぞれ、式（Ｂ－１）における○および●と同じ配向である。）である。〕から水素原子を一つ除いた基である。式（Ｂ－２’）におけるｓの定義、例および好ましい例は、式（Ｂ－２）のものと同様である。したがって、式（Ｂ－２’）では、ＶおよびＶ’の一方が、－Ｎ（－）－、または－ＣＨ（－）－であってもよい。あるいは、式（Ｂ－２’）では、Ｖ１は、上記（ａ）または（ｂ）の２価の基から水素原子が一つ除かれた３価の基であってもよい。あるいは、式（Ｂ－２’）では、ｓ個の－ＣＨ_２－のうちの一つが－ＣＨ（－）－であってもよい。 The group obtained by removing one hydrogen atom from the above formula (B-2) is represented by the following formula (B-2′):

(In the formula,
V and V′ are the same or different and are —NH—, —O—, —CH ₂ —, or a single bond;
V is (a) a divalent group containing a bioorthogonal functional group or (b) a divalent group without a bioorthogonal functional group;
s is any integer from 0 to 10,
○ and ● in formula (B-2′) have the same orientations as ○ and ● in formula (B-1), respectively. ). ] with one hydrogen atom removed. The definition, examples and preferred examples of s in formula (B-2') are the same as those in formula (B-2). Therefore, in formula (B-2'), one of V and V' may be -N(-)- or -CH(-)-. Alternatively, in formula (B-2′), V1 may be a trivalent group obtained by removing one hydrogen atom from the divalent group (a) or (b) above. Alternatively, in formula (B-2'), one of the s -CH ₂ - may be -CH(-)-.

〔式中、
Ａ、Ｒ、Ｃ、Ｆ１、Ｆ２、ｐ、ｐ’、ｑ、ｑ’、Ｘ、Ｘ’、Ｒ_１ａ、Ｒ_１ｂ、Ｒ_１ａ’、Ｒ_１ｂ’、Ｙ、Ｙ’、ＺおよびＺ’の定義、例および好ましい例は、上述したものと同じである。〕 In this case, the above formula (III) can be defined as the following formula (III'').

[In the formula,
Definitions of A, R, C, F1, F2, p, p', q, q', X, X', R _1a , R _1b , R _1a' , R _1b' , Y, Y', Z and Z' , examples and preferred examples are the same as described above. ]

In the above formulas (LB′(F)′) and (III″), the length from C (carbon atom) at C=Z to C (carbon atom) at C=Z′ is A and It is the same as the length of the main chain connecting Rs. In these formulas, the length from C in C=Z to C in C=Z' also refers to the connecting chain of the substructure connecting C in C=Z to C in C=Z' (hydrogen atoms and substituents ) can also be defined as the number of atoms constituting The number of such atoms is the same as the number of atoms constituting the main chain connecting A and R. The connecting chain of the partial structure connecting C in C=Z to C in C=Z' (excluding hydrogen atoms and substituents) may or may not contain a ring structure, but may contain a ring structure. preferably not included. The linking chain (excluding hydrogen atoms and substituents) may preferably be free of peptide moieties.

3-5. Binding sites of partial structures other than soluble proteins possessed by soluble proteins (regioselectivity)
A partial structure other than a soluble protein (eg, ALB'(-F)-R') can be regioselectively bound to the target region as described above in the soluble protein (T). Specifically, T contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and the specific amino acid residue in a non-target region other than the target region When it contains 5 or more amino acid residues, the partial structure other than the soluble protein can bind to one or more specific amino acid residues contained in the target region with a position selectivity of 30% or more. . Definitions, examples and preferred examples of target regions and regioselectivity are provided above.

3-6. Number of partial structures other than soluble protein possessed by soluble protein The number of partial structures other than soluble protein possessed by soluble protein (T) (e.g., ALB'(-F)-R') varies. can do. For example, if T is a multimeric protein comprising a plurality of monomeric proteins, T can have substructures other than T in the corresponding plurality of target regions in the plurality of monomeric proteins. . As a result, T can have multiple partial structures other than T. Therefore, the structure represented by formula (III), (III'), or (III'') indicates that T may have one or more partial structures other than T. The number of partial structures other than T that T has can be adjusted by appropriately setting conditions such as the type of soluble protein and the reaction ratio between the soluble protein and structural units introduced therein. Such number may vary depending on the type of soluble protein, but may be, for example, 1 to 8, preferably 1 to 4, more preferably 1 or 2.

In certain embodiments, if the soluble protein is a multimeric protein composed of a plurality of monomeric proteins, the number of partial structures other than the soluble protein possessed by the soluble protein may be more than one. good. According to the present invention, a plurality of partial structures other than soluble proteins can be introduced into the same target region of a plurality of monomeric proteins.

In preferred embodiments, the soluble protein may be an antibody comprising multiple heavy chains. Definitions, examples and preferred examples of antibodies are given above. In the present invention, the number of partial structures other than antibodies possessed by antibodies is 1 or 2 (preferably 2) for IgG, IgE, and IgD, and 1 to 4 (preferably 4), and for IgM may be 1 to 8 (preferably 8).

3-7. Methods of Production The present invention provides methods of producing a conjugate or salt thereof having an affinity agent for a soluble protein, a cleavable moiety, a functional agent and a soluble protein, including:
(B1) A conjugate having an affinity substance for a soluble protein and a soluble protein containing a cleavable portion or a salt thereof with a functional substance to obtain a soluble protein-affinity substance, a cleavable portion, a functional substance and a soluble protein To produce the body or its salts.

The affinity agent for the soluble protein and the soluble protein comprising the cleavable moiety are represented by formula (II), preferably formula (II'), more preferably formula (II''). A conjugate comprising an affinity agent for a soluble protein, a cleaving moiety, a functional agent and a soluble protein is represented by formula (III), preferably formula (III'), more preferably formula (III'').

Affinity agents for soluble proteins, and soluble proteins or salts thereof that include cleavable moieties have bioorthogonal functional groups that can react with functional agents, and thus can react with functional agents. Such a reaction can be appropriately carried out under conditions (mild conditions) that do not cause protein denaturation/degradation (e.g., cleavage of amide bonds), as described in "2-6. Production method" above. can.

In the reaction system, the molar ratio (Y/X) of the affinity substance for the soluble protein and the functional substance (Y) to the soluble protein containing the cleavable moiety or salt thereof (X) is determined by the soluble protein (e.g., antibody) and Although it is not particularly limited because it varies depending on the type of functional substance, the number of sites in the soluble protein to be modified by the functional substance (eg, DAR), etc., it is, for example, 0.1 to 100, preferably 0.5 to 80, more preferably 1 to 70, even more preferably 2 to 50, and particularly preferably 3 to 30.

Confirmation of the production of a conjugate having an affinity substance for a soluble protein, a cleavable moiety, a functional substance and a conjugate having a soluble protein or a salt thereof, depends on the specific starting material and the molecular weight of the product, but for example by electrophoresis. , chromatography (eg, gel filtration chromatography, ion exchange chromatography, reversed-phase column chromatography, HPLC), or mass spectrometry, preferably mass spectrometry. Confirmation of regioselectivity, confirmation of the number of partial structures other than the soluble protein, and purification can be carried out as appropriate by the method described in "2-6. Production method" above.

The present invention also provides a method for producing a conjugate or salt thereof having an affinity agent for a soluble protein, a cleavable moiety, a functional agent and a soluble protein, comprising:
(B2) reacting a compound or a salt thereof containing an affinity substance for a soluble protein, a cleavable moiety and a reactive group with the soluble protein to produce an affinity substance for the soluble protein and a soluble protein containing the cleavable moiety or a salt thereof; and (B3) reacting the affinity agent for the soluble protein and the soluble protein comprising the cleavable moiety or a salt thereof with the functional agent to produce the affinity agent for the soluble protein, the cleavable moiety, the functional agent and Producing a conjugate with a soluble protein or a salt thereof.

The above step (B2) can be performed in the same manner as the above step (A1) described in "2-6. Production method" above. The step (B3) above can be performed in the same manner as the step (B1) above. The steps (B2) and (B3) above can be performed separately. Alternatively, the above steps (B2) and (B3) are carried out by a reaction pair between a specific amino acid residue that is a reaction target in a soluble protein and a reactive group, and a functional group in a functional substance and a bioorthogonal functional group. can be performed simultaneously, depending on the combination of reaction pairs with

4. Soluble protein or salt thereof having bioorthogonal functional group 4-1. Outline of Production Method The present invention provides a method for producing a soluble protein having a bioorthogonal functional group represented by formula (IV) or a salt thereof.
L1-B-R'-T (IV)
[In the formula,
L1 is (i′) a monovalent group containing a bioorthogonal functional group or (ii′) a monovalent group not containing a bioorthogonal functional group;
B is (a) a divalent group that includes a bioorthogonal functional group or (b) a divalent group that does not include a bioorthogonal functional group;
R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein. ]

4-2. (i′) a monovalent group containing a bioorthogonal functional group or (ii′) a monovalent group (L1) containing no bioorthogonal functional group
In formula (IV), L1 is (i') a monovalent group that includes a bioorthogonal functional group or (ii') a monovalent group that does not include a bioorthogonal functional group. Such monovalent groups are monovalent groups that can be generated by cleavage of a cleavable linker, which is a divalent group containing a cleavable moiety as described above. Thus, L1 may be a residue of a cleavable moiety as described above or a monovalent group that can be generated by cleavage of a chemical structure. More specifically, (i′) a monovalent group containing a bioorthogonal functional group is a cleavable moiety having the ability to generate a bioorthogonal functional group on the reactive group side by cleavage as described above (i). It may be a monovalent group that can be generated by cleavage of a cleavable linker that is a bivalent group containing (ii′) a monovalent group that does not contain a bioorthogonal functional group, (ii) bio It may be a monovalent group that can be generated by cleavage of a cleavable linker, which is a divalent group containing a cleavable moiety that does not have the ability to generate an orthogonal functional group on the reactive group side.

In certain embodiments, L1 may be represented by any one of formula (L1-1) or (L1-2) below:
C1-Lb (L1-1)
C1 (L1-2)
[In the formula,
Lb is a divalent group,
C1 is a bioorthogonal functional group or a group other than a bioorthogonal functional group. ]

As the divalent group, for example, a divalent hydrocarbon group which may have a substituent, a divalent heterocyclic group which may have a substituent, -C (= O) -, - NR _a — (R _a represents a hydrogen atom or a substituent), —O—, —S—, —C(═S)—, and two or more of these (for example, 2 to 8, preferably 2 to 6, Groups consisting of combinations of 2 to 4) are more preferred. Definitions, examples, and preferred examples of the divalent hydrocarbon group, the divalent heterocyclic group, and the substituent are as described above.

Definitions, examples and preferred examples of bioorthogonal functional groups are given above.

Examples of the groups other than the bioorthogonal functional groups include, among the substituents described above, those other than the bioorthogonal functional groups. Such groups other than bioorthogonal functional groups include, for example, alkyl, cycloalkyl, aralkyl, monovalent heterocyclic groups, hydroxyl, amino, alkyloxy (alkoxy), cycloalkyloxy, aralkyloxy). . Definitions, examples and preferred examples of these groups and constituents of these groups (e.g., alkyl in alkyloxy (alkoxy), cycloalkyl in cycloalkyloxy, and aralkyl in aralkyloxy) are the same as those described above. be.

〔式中、
ｐは、０～１０の任意の整数であり、
ｑは、０～１０の任意の整数であり、
Ｘは、炭素原子、窒素原子、または単結合（ここで、Ｘが窒素原子である場合、Ｒ_１ｂは存在しない。Ｘが単結合である場合、Ｒ_１ａおよびＲ_１ｂは存在しない）であり、
Ｒ_１ａ、およびＲ_１ｂは、同一または異なって、水素原子、または上述する置換基からなる群より選ばれる。
○（白丸）は、Ｃ１に対する結合を示し、●（黒丸）はＢに対する結合を示す。〕で表されてもよい。 In certain embodiments, Lb is (Lb'):

[In the formula,
p is any integer from 0 to 10,
q is any integer from 0 to 10,
X is a carbon atom, a nitrogen atom, or a single bond (wherein when X is a nitrogen atom, R _1b is absent; when X is a single bond, R _1a and R _1b are absent);
R _1a and R _1b are the same or different and are selected from the group consisting of a hydrogen atom or the substituents described above.
O (white circle) indicates binding to C1 and ● (filled circle) indicates binding to B. ] may be represented.

Definitions, examples and preferred examples of p, q and X, and R _1a and R _1b (substituents) are as described above.

4-3. Partial structure "L1-B"
4-3-1. Length of partial structure "L1-B" connecting L1 terminal portion and R' B”) length (or bioorthogonal functional groups in soluble proteins or salts thereof regioselectively having bioorthogonal functional groups, not limited to specific formulas such as formula (IV) and the number of atoms in the main chain connecting the side chains of specific amino acid residues; It can be designed as appropriate according to various factors as described in .

The length of the partial structure connecting the L1 terminal portion (or C1 terminal portion) generated by cleavage and R' may be, for example, about 2.5 Å or more, preferably about 4 Å or more, more preferably about 5 Å or more. . Substructure lengths may also be, for example, about 15 Å or less, preferably about 12 Å or less, more preferably about 8 Å or less. More specifically, the substructure length may be, for example, about 2.5-15 Å, preferably about 4-12 Å, more preferably about 5-8 Å.

More specifically, the length of the partial structure connecting the L1 terminal portion (or C1 terminal portion) and R′ generated by cleavage is the atoms constituting the connecting chain of the partial structure (excluding hydrogen atoms and substituents) It can also be specified as a number. The number of atoms constituting the linking chain may be, for example, 2 (about 2.5 Å) or more, preferably 3 (about 4 Å) or more, more preferably 4 (about 5 Å) or more. The number of atoms constituting the linking chain may also be, for example, 10 (about 15 Å) or less, preferably 8 (about 12 Å) or less, more preferably 6 (about 8 Å) or less. More specifically, the number of atoms constituting the connecting chain may be, for example, 2-10, preferably 3-8, more preferably 4-6.

When the connecting chain of the partial structure connecting the L1 terminal portion (or C1 terminal portion) and R' generated by cleavage is a chain structure that does not contain a divalent ring structure, the number of atoms in the connecting chain is can be determined by counting the number of atoms of

On the other hand, when the connecting chain of the partial structure connecting the L1 terminal portion (or the C1 terminal portion) and R′ generated by cleavage has a structure containing a divalent ring structure, the number of atoms of the connecting chain and the length described above are Not necessarily corresponding, the length that can be defined by the number of atoms in the linking chain tends to be shorter than the lengths mentioned above. Even in such a case, the number of atoms in the linking chain can be conveniently counted from the viewpoint that the length of the linking chain is defined by the number of atoms. Specifically, the number of atoms in the linking chain in such a case is, as described above, in addition to the number of atoms in the chain structure that does not contain a divalent ring structure in the linking chain, may be determined by counting the number of atoms in the shortest path connecting two bonds of .

Preferably, the linking chain of the partial structure linking the L1 terminal portion (or C1 terminal portion) and R' generated by cleavage may be a chain structure that does not contain a divalent ring structure. In this case, L1-B can be appropriately designed so that the connecting chain of the partial structure connecting the terminal portion and R' generated by cleavage does not contain a divalent cyclic group.

In certain embodiments, the substructure represented by L1-B preferably does not contain peptide moieties. In this case, the soluble protein having the functional substance of the present invention (eg, antibody-drug conjugate) obtained using the soluble protein having the bioorthogonal functional group of the present invention contains a peptide portion that can have immunogenicity. It has the advantage that it cannot be included as a linker.

4-3-2. Specific Structure of Partial Structure “L1-B” In the above formula (IV), L1 and B are structures that can be associated with each other. Therefore, in the above formula (IV), L1 and B can be defined as a partial structure represented by "L1-B".

In certain embodiments, when L is (i′) a monovalent group comprising a bioorthogonal functional group, B is (a) a divalent group comprising a bioorthogonal functional group, or (b) a bio It is a divalent group that does not contain an orthogonal functional group.

Preferably, when L1 is (i') a monovalent group comprising a bioorthogonal functional group, B is (a) a divalent group comprising a bioorthogonal functional group. In this case, the bioorthogonal functional group in (i') may be the same as or different from the bioorthogonal functional group in (a). From the viewpoint of adopting a simpler structure and/or improving reactivity with a single functional substance, the bioorthogonal functional group in (i′) is the same as the bioorthogonal functional group in (a). There may be. On the other hand, from the viewpoint of ensuring differentiated reactivity with two or more functional substances, and not using some bioorthogonal functional groups in the reaction, the bioorthogonal functional group in (i′) is It may be different from the bioorthogonal functional group in (a).

Alternatively, when L1 is (i') a monovalent group containing a bioorthogonal functional group, B may be (b) a divalent group containing no bioorthogonal functional group. In this case, the soluble protein having a bioorthogonal functional group represented by formula (IV) or a salt thereof has a simpler structure and is therefore easier to synthesize.

In another particular embodiment, when L1 is (ii') a monovalent group that does not contain a bioorthogonal functional group, then B is (a) a divalent group that contains a bioorthogonal functional group.

〔式中、
Ｃ１、ｐ、ｑ、Ｘ、Ｒ_１ａ、Ｒ_１ｂ、Ｙ、およびＺの定義、例および好ましい例は、上述したものと同じであり、
●（黒丸）は、Ｒ’に対する結合を示す。〕 In certain embodiments, the structural unit represented by L1-B may be represented by the following formula (L1B').

[In the formula,
The definitions, examples and preferred examples of C1, p, q, X, R _1a , R _1b , Y and Z are the same as described above,
● (filled circle) indicates a bond to R′. ]

〔式中、
Ｃ１、ｐ、ｑ、Ｘ、Ｒ_１ａ、Ｒ_１ｂ、Ｙ、Ｚ、Ｒ’、およびＴの定義、例および好ましい例は、上述したものと同じである。〕 Therefore, the above formula (IV) can be defined as the following formula (IV').

[In the formula,
The definitions, examples and preferred examples of C1, p, q, X, R _1a , R _1b , Y, Z, R' and T are the same as described above. ]

In the above formulas (L1B') and (IV'), the length from C to C1 in C=Z is the length of the partial structure connecting the L1 terminal portion (or C1 terminal portion) generated by cleavage and R'. It is the same. In these formulas, the length from C to C in C=Z is also defined as the number of atoms constituting the connecting chain (excluding hydrogen atoms and substituents) of the substructure from C to C in C=Z can also The number of such atoms is the same as the number of atoms constituting the partial structure (excluding hydrogen atoms and substituents) connecting the L1 terminal portion (or C1 terminal) generated by cleavage and R'. The connecting chain (excluding hydrogen atoms and substituents) of the partial structure connecting C to C1 in C=Z may or may not contain a ring structure, but may not contain a ring structure. preferable. The linking chain (excluding hydrogen atoms and substituents) may preferably be free of peptide moieties.

4-4. Binding sites of partial structures other than soluble proteins possessed by soluble proteins (regioselectivity)
Partial structures other than soluble proteins (eg, L1-BR') can be regioselectively bound to target regions as described above in soluble proteins (T). Specifically, T contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and the specific amino acid residue in a non-target region other than the target region When it contains 5 or more amino acid residues, the partial structure other than the soluble protein can bind to one or more specific amino acid residues contained in the target region with a position selectivity of 30% or more. . Definitions, examples and preferred examples of target regions and regioselectivity are provided above.

4-5. Number of partial structures other than soluble protein possessed by soluble protein The number of partial structures (eg, L1-BR') possessed by the soluble protein (T) other than the soluble protein can vary. For example, if T is a multimeric protein comprising a plurality of monomeric proteins, T can have substructures other than T in the corresponding plurality of target regions in the plurality of monomeric proteins. . As a result, T can have multiple partial structures other than T. Therefore, the structure represented by formula (IVI), (IV'), or IV'') indicates that T may have one or more partial structures other than T. The number of partial structures other than T that T has can be adjusted by appropriately setting conditions such as the type of soluble protein and the reaction ratio between the soluble protein and structural units introduced therein. Such number may vary depending on the type of soluble protein, but may be, for example, 1 to 8, preferably 1 to 4, more preferably 1 or 2.

In certain embodiments, if the soluble protein is a multimeric protein composed of a plurality of monomeric proteins, the number of partial structures other than the soluble protein possessed by the soluble protein may be more than one. good. According to the present invention, a plurality of partial structures other than soluble proteins can be introduced into the same target region of a plurality of monomeric proteins.

In preferred embodiments, the soluble protein may be an antibody comprising multiple heavy chains. Definitions, examples and preferred examples of antibodies are given above. In the present invention, the number of partial structures other than antibodies possessed by antibodies is 1 or 2 (preferably 2) for IgG, IgE, and IgD, and 1 to 4 (preferably 4), and for IgM may be 1 to 8 (preferably 8).

4-6. Methods of Making The present invention provides methods of making soluble proteins or salts thereof having bioorthogonal functional groups, including:
(C1) Cleavage of a soluble protein or salt thereof comprising an affinity substance for a soluble protein and a cleavable moiety to produce a soluble protein or salt thereof having a bioorthogonal functional group.

The affinity agent for soluble proteins and the soluble protein comprising the cleavable moiety is represented by formula (II), preferably formula (II'), more preferably formula (II''). Soluble proteins with bioorthogonal functional groups are represented by formula (IV), preferably formula (IV'), more preferably formula (IV'').

A soluble protein having an affinity for a soluble protein and a soluble protein containing a cleavable moiety or a salt thereof can be cleaved because it has a cleavable moiety that can be cleaved by the cleavage treatment as described above. Such a cleavage reaction should be appropriately carried out under conditions (mild conditions) that do not cause protein denaturation/degradation (e.g., amide bond cleavage) as described in "2-6. Production method" above. can be done.

Examples of the cleavage treatment include (a) treatment with one or more substances selected from the group consisting of acidic substances, basic substances, reducing agents, oxidizing agents, and enzymes; (c) leaving when using a cleavable linker comprising an autolytic cleavable moiety. Conditions for such cutting treatment are common technical knowledge in the art (eg, G. Leriche, L. Chisholm, A. Wagner, Bioorganic & Medicinal Chemistry. 20, 571 (2012); Feng P. et al., Journal of American Chemical Society.132, 1500 (2010).;
et al. , Journal of Controlled Release, 99, 423 (2004). DeSimone, J.; M. , Journal of American Chemical Society. 132, 17928 (2010); Thompson, D.; H. , Journal of Controlled Release, 91, 187 (2003); G. , Journal of Controlled Release, 95, 291 (2004)).

Confirmation of the production of soluble proteins or salts thereof with bioorthogonal functional groups, depending on the specific starting material and the molecular weight of the product, can be performed, for example, by electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reversed-phase column chromatography, HPLC), or mass spectrometry, preferably mass spectrometry. Confirmation of regioselectivity, confirmation of the number of partial structures other than the soluble protein, and purification can be carried out as appropriate by the method described in "2-6. Production method" above.

The present invention also provides a method for producing a soluble protein or salt thereof having a bioorthogonal functional group, comprising:
(C2) reacting a compound or a salt thereof containing an affinity substance for a soluble protein, a cleavable moiety and a reactive group with a soluble protein to produce an affinity substance for the soluble protein and a soluble protein containing the cleavable moiety or a salt thereof; and (C3) cleaving the soluble protein or salt thereof comprising an affinity substance for the soluble protein and the cleavable moiety to produce a soluble protein or salt thereof having a bioorthogonal functional group. matter.

The above step (C2) can be performed in the same manner as the above step (A1) described in "2-6. Production method" above. The step (C3) can be performed in the same manner as the step (C1). The above steps (C2) and (C3) can be performed separately. Alternatively, steps (C2) and (C3) above can be performed simultaneously, depending on factors such as the combination of reactive groups and bioorthogonal functional groups that can be generated by cleavage (non-reactive).

5. Method for producing a soluble protein having a functional substance or a salt thereof 5-1. Overview The present invention provides a method for producing a soluble protein having a functional substance represented by formula (V) or a salt thereof.
F-(L1-B)'-R'-T (V)
[In the formula,
L1 is (i′) a monovalent group containing a bioorthogonal functional group or (ii′) a monovalent group not containing a bioorthogonal functional group;
B is (a) a divalent group that includes a bioorthogonal functional group or (b) a divalent group that does not include a bioorthogonal functional group;
The structural unit represented by (L1-B)' includes a moiety generated by a reaction between a functional substance and either one or both of the bioorthogonal functional groups in (i') and (a). is a divalent structural unit,
F is a functional substance,
R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein. ]

5-2. Partial structure "F-(L1-B)'"
5-2-1. Length of partial structure "L1-B" connecting F and R' length (or main linking side chain of a functional substance and a specific amino acid residue in a soluble protein or a salt thereof regioselectively having a functional substance, which is not limited to a specific formula such as formula (V) The number of atoms in the chain, or if the functional agent is attached to a soluble protein via a bioorthogonal functional group, the atoms of the backbone linking the bioorthogonal functional group and the side chain of a particular amino acid residue. hereinafter the same) is appropriately designed according to various factors as described in the above “1-6-1. Length of the main chain in the partial structure “LB” connecting A and R” can do.

The length of the partial structure connecting F and R' may be, for example, about 2.5 Å or longer, preferably about 4 Å or longer, more preferably about 5 Å or longer. Substructure lengths may also be, for example, about 15 Å or less, preferably about 12 Å or less, more preferably about 8 Å or less. More specifically, the substructure length may be, for example, about 2.5-15 Å, preferably about 4-12 Å, more preferably about 5-8 Å.

More specifically, the length of the partial structure connecting F and R' can also be defined as the number of atoms constituting the connecting chain (excluding hydrogen atoms and substituents) of the partial structure. The number of atoms constituting the linking chain may be, for example, 2 (about 2.5 Å) or more, preferably 3 (about 4 Å) or more, more preferably 4 (about 5 Å) or more. The number of atoms constituting the connecting chain is also, for example, 1
It may be 0 (about 15 Å) or less, preferably 8 (about 12 Å) or less, more preferably 6 (about 8 Å) or less. More specifically, the number of atoms constituting the linking chain may be, for example, 2-10, preferably 3-8, more preferably 4-6.

When the connecting chain of the partial structure connecting F and R' is a chain structure that does not contain a divalent ring structure, the number of atoms in the connecting chain can be determined by counting the number of atoms in the connecting chain. .

On the other hand, when the connecting chain of the partial structure connecting F and R' is a structure containing a divalent ring structure, the number of atoms of the connecting chain does not necessarily correspond to the length described above, and is defined by the number of atoms of the connecting chain. The lengths that can be used tend to be shorter than the lengths mentioned above. Even in such a case, the number of atoms in the linking chain can be conveniently counted from the viewpoint that the length of the linking chain is defined by the number of atoms. Specifically, the number of atoms in the linking chain in such a case is, as described above, in addition to the number of atoms in the chain structure that does not contain a divalent ring structure in the linking chain, may be determined by counting the number of atoms in the shortest path connecting two bonds of .

Preferably, the connecting chain of the partial structure connecting F and R' may be a chain structure that does not contain a divalent ring structure. L1-B can be appropriately designed so that the connecting chain of the partial structure connecting F and R' does not contain a divalent cyclic group.

In certain embodiments, the substructure represented by L1-B preferably does not contain peptide moieties. In this case, the soluble protein (eg antibody drug conjugate) with the functional substance of the invention has the advantage that it does not contain a potentially immunogenic peptide moiety as a linker.

5-2-2. Specific Structure of Partial Structure “F-(L1-B)′” In formula (V), L1 and B in F-(L1-B)′ are structures that can be associated with each other. Therefore, in the above formula (V), L1 and B can be defined as a partial structure represented by "L1-B".

Further, the structural unit represented by F-(L1-B)' is generated by reaction between the functional substance and either one or both of the bioorthogonal functional groups in (i') and (a). This is the part to do. A bioorthogonal functional group subjected to reaction is absent in formula (V). Thus, in formula (V) either or both of the bioorthogonal functional groups in (i') and (a) are absent. The moiety generated by the reaction between the functional agent and either or both of the bioorthogonal functional groups in (i′) and (a) is the reaction between the functional agent and the bioorthogonal functional group is the same as the part generated by Therefore, herein, examples of moieties generated by reaction between a functional substance and either one or both of the bioorthogonal functional groups in (i′) and (a) (names of residues) and Specific examples (chemical structural formulas) are the same as those described above for moieties generated by reactions between functional substances and bioorthogonal functional groups.

In one embodiment, when L1 is (i′) a monovalent group comprising a bioorthogonal functional group, B is (a) a divalent group comprising a bioorthogonal functional group, or (b) a bioorthogonal It is a divalent group that does not contain a sexual functional group.

In a preferred embodiment, when L1 is (i') a monovalent group comprising a bioorthogonal functional group, B is (a) a divalent group comprising a bioorthogonal functional group. In this case, the bioorthogonal functional group in (i′) may be the same or different from the bioorthogonal functional group in (a). From the viewpoint of adopting a simpler structure and/or improving reactivity with a single functional substance, the bioorthogonal functional group in (i′) is the same as the bioorthogonal functional group in (a). There may be. On the other hand, from the viewpoint of ensuring differentiated reactivity with two or more functional substances and not using some bioorthogonal functional groups in the reaction, the bioorthogonal functional group in (i′) is It may be different from the bioorthogonal functional group in (a).

In another preferred embodiment, when L is (i′) a monovalent group containing a bioorthogonal functional group, B may be (b) a divalent group that does not contain a bioorthogonal functional group. good. In this case, the soluble protein having a functional substance represented by formula (V) or a salt thereof has a simpler structure, and is easy to synthesize.

In another embodiment, when L1 is (ii') a monovalent group that does not contain a bioorthogonal functional group, B is (a) a divalent group that contains a bioorthogonal functional group.

In certain embodiments, when the bioorthogonal functional groups in (i′) and (a) are different, the soluble protein or salt thereof having a functional agent represented by formula (V) is represented by formula (V1 ) or (V2).

L1-B'(-F)-R'-T (V1)
[In the formula,
L1 is (i′) a monovalent group containing a bioorthogonal functional group or (ii′) a monovalent group not containing a bioorthogonal functional group;
B' is a divalent group comprising a moiety generated by a reaction between a functional agent and a bioorthogonal functional group;
F is a functional substance,
R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein. ]

FL1'-B-R'-T (V2)
[In the formula,
L1' is a divalent group comprising a moiety produced by a reaction between a functional agent and (i') a monovalent group comprising a bioorthogonal functional group;
B is (a) a divalent group that includes a bioorthogonal functional group or (b) a divalent group that does not include a bioorthogonal functional group;
F is a functional substance,
R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein. ]

L1' is a divalent group that includes a moiety produced by a reaction between a functional agent and (i') a monovalent group that includes a bioorthogonal functional group. The moiety generated by the reaction between the functional substance and (i′) a monovalent group containing a bioorthogonal functional group is the moiety generated by the reaction between the functional substance and the bioorthogonal functional group. are the same. Therefore, in the present specification, examples (residue name) and specific examples (chemical structure Formula ) is the same as described above for moieties generated by reactions between functional agents and bioorthogonal functional groups. Therefore, since the functional agent reacts with the bioorthogonal functional group, a divalent functional agent comprising a moiety produced by reaction between the functional agent and (i′) a monovalent group comprising a bioorthogonal functional group. The group (L1') can also be expressed as a divalent group including a moiety generated by a reaction between a functional substance and a bioorthogonal functional group (same below). Definitions, examples and preferred examples of the divalent group including the portion generated by the reaction between the functional substance and the bioorthogonal functional group in L1' are the functional substance and the bioorthogonal functional group in B' It may be the same as that of a divalent group including a moiety generated by a reaction between and.

In another specific embodiment, when the bioorthogonal functional groups in (i') and (a) are the same or different, the soluble protein having a functional agent represented by formula (V) or a salt thereof is , may be represented by the formula (V3).

Fa-L1'-B'(-Fb)-R'-T (V3)
[In the formula,
L1' is a divalent group comprising a moiety produced by a reaction between a functional agent and (i') a monovalent group comprising a bioorthogonal functional group;
B' is a divalent group that includes a moiety generated by a reaction between a functional agent and a bioorthogonal functional group;
Fa and Fb are the same or different functional substances, respectively;
R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein. ]

〔式中、
Ｆ、Ｃ１、ｐ、ｑ、Ｘ、Ｒ_１ａ、Ｒ_１ｂ、およびＺの定義、例および好ましい例は、上述したものと同じであり、
Ｙ’は、前記式（Ｂ－１）のＹから水素原子を一つ除いた残基であり、
●（黒丸）は、Ｒ’に対する結合を示す。〕 In certain embodiments, the structural unit represented by L1-B'(-F) in formula (V1) above may be represented by formula (L1B'(F)) below.

[In the formula,
The definitions, examples and preferred examples of F, C1, p, q, X, R _1a , R _1b and Z are the same as described above,
Y' is a residue obtained by removing one hydrogen atom from Y in the formula (B-1),
● (filled circle) indicates a bond to R′. ]

The definition, examples and preferred examples of the residue obtained by removing one hydrogen atom from Y in formula (B-1) are the same as those described above.

〔式中、
Ｆ、Ｃ１、ｐ、ｑ、Ｘ、Ｒ_１ａ、Ｒ_１ｂ、Ｚ、Ｒ’、およびＴの定義、例および好ましい例は、上述したものと同じであり、
Ｙ’は、前記式（Ｂ－１）のＹから水素原子を一つ除いた残基である。〕 Therefore, the above formula (V1) can be defined as the following formula (V1').

[In the formula,
The definitions, examples and preferred examples of F, C1, p, q, X, R _1a , R _1b , Z, R′, and T are the same as described above,
Y' is a residue obtained by removing one hydrogen atom from Y in the above formula (B-1). ]

〔式中、
Ｆ、ｐ、ｑ、Ｘ、Ｒ_１ａ、Ｒ_１ｂ、Ｙ、およびＺの定義、例および好ましい例は、上述したものと同じであり、
Ｃ１’は、機能性物質と生体直交性官能基との間の反応により生成する部分であり、
●（黒丸）は、Ｒ’に対する結合を示す。〕 In certain embodiments, the structural unit represented by FL1′-B in formula (V2) above may be represented by formula (FL1′B) below.

[In the formula,
The definitions, examples and preferred examples of F, p, q, X, R _1a , R _1b , Y and Z are the same as described above,
C1' is a moiety generated by a reaction between a functional substance and a bioorthogonal functional group;
● (filled circle) indicates a bond to R′. ]

C1' is the moiety generated by the reaction between the functional agent and the bioorthogonal functional group. The definition, examples and preferred examples of the portion generated by the reaction between the functional substance and the bioorthogonal functional group in C1′ are described in the above “3-3. Between the functional substance and the bioorthogonal functional group divalent group (B′) containing a moiety generated by reaction” (the same shall apply hereinafter).

〔式中、
Ｆ、ｐ、ｑ、Ｘ、Ｒ_１ａ、Ｒ_１ｂ、Ｙ、Ｚ、Ｒ’、およびＴの定義、例および好ましい例は、上述したものと同じであり、
Ｃ１’は、機能性物質と生体直交性官能基との間の反応により生成する部分である。〕 Therefore, the above formula (V2) can be defined as the following formula (V2').

[In the formula,
The definitions, examples and preferred examples of F, p, q, X, R _1a , R _1b , Y, Z, R′ and T are the same as described above,
C1' is the moiety generated by the reaction between the functional substance and the bioorthogonal functional group. ]

〔式中、
Ｆａ、Ｆｂ、ｐ、ｑ、Ｘ、Ｒ_１ａ、Ｒ_１ｂ、およびＺの定義、例および好ましい例は、上述したものと同じであり、
Ｃ１’は、機能性物質と生体直交性官能基との間の反応により生成する部分であり、
Ｙ’は、前記式（Ｂ－１）のＹから水素原子を一つ除いた残基であり、
●（黒丸）は、Ｒ’に対する結合を示す。〕 In certain embodiments, the structural unit represented by Fa-L1'-B'(-Fb) in formula (V3) above may be represented by the following formula (FaL1'B'(Fb)).

[In the formula,
The definitions, examples and preferred examples of Fa, Fb, p, q, X, R _1a , R _1b and Z are the same as described above,
C1' is a moiety generated by a reaction between a functional substance and a bioorthogonal functional group;
Y' is a residue obtained by removing one hydrogen atom from Y in the formula (B-1),
● (filled circle) indicates a bond to R′. ]

〔式中、
Ｆａ、Ｆｂ、ｐ、ｑ、Ｘ、Ｒ_１ａ、Ｒ_１ｂ、Ｚ、Ｒ’、およびＴの定義、例および好ましい例は、上述したものと同じであり、
Ｃ１’は、機能性物質と生体直交性官能基との間の反応により生成する部分であり、
Ｙ’は、前記式（Ｂ－１）のＹから水素原子を一つ除いた残基である。〕 Therefore, the above formula (V3) can be defined as the following formula (V3').

[In the formula,
The definitions, examples and preferred examples of Fa, Fb, p, q, X, R _1a , R _1b , Z, R′ and T are the same as described above,
C1' is a moiety generated by a reaction between a functional substance and a bioorthogonal functional group;
Y' is a residue obtained by removing one hydrogen atom from Y in the above formula (B-1). ]

In the above formulas (L1B'(F)), (FL1'B), and (FaL1'B'(F2)), and (V1'), (V2') and (V3'), from C at C=Z The length up to C1 is the same as the length of the partial structure connecting the L1 terminal portion (or C1 terminal portion) generated by cleavage and R'. In these formulas, the length from C to C in C=Z is also defined as the number of atoms constituting the connecting chain (excluding hydrogen atoms and substituents) of the substructure from C to C in C=Z can also The number of such atoms is the same as the number of atoms constituting the partial structure (excluding hydrogen atoms and substituents) connecting the L1 terminal portion (or C1 terminal) generated by cleavage and R'. The connecting chain (excluding hydrogen atoms and substituents) of the partial structure connecting C to C1 in C=Z may or may not contain a ring structure, but may not contain a ring structure. preferable. The linking chain (excluding hydrogen atoms and substituents) may preferably be free of peptide moieties.

5-3. Binding sites of partial structures other than soluble proteins possessed by soluble proteins (regioselectivity)
A partial structure other than a soluble protein (eg, F-(L1-B)'-R') can be regioselectively bound to the target region as described above in the soluble protein (T). Specifically, T contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and the specific amino acid residue in a non-target region other than the target region When it contains 5 or more amino acid residues, the partial structure other than the soluble protein can bind to one or more specific amino acid residues contained in the target region with a position selectivity of 30% or more. . Definitions, examples and preferred examples of target regions and regioselectivity are provided above.

5-4. Number of partial structures other than soluble protein possessed by soluble protein The number of partial structures other than soluble protein possessed by soluble protein (T) (e.g., F-(L1-B)'-R') may vary. can be done. For example, if T is a multimeric protein comprising a plurality of monomeric proteins, T can have substructures other than T in the corresponding plurality of target regions in the plurality of monomeric proteins. . As a result, T can have multiple partial structures other than T. Therefore, structures represented by formulas (V), (V1), (V2), (V3), (V1′), (V2′), or (V3′) are those in which T is a partial structure other than T Indicates that one or more may be provided. The number of partial structures other than T that T has can be adjusted by appropriately setting conditions such as the type of soluble protein and the reaction ratio between the soluble protein and structural units introduced therein. Such number may vary depending on the type of soluble protein, but may be, for example, 1 to 8, preferably 1 to 4, more preferably 1 or 2.

In certain embodiments, if the soluble protein is a multimeric protein composed of a plurality of monomeric proteins, the number of partial structures other than the soluble protein possessed by the soluble protein may be more than one. good. According to the present invention, a plurality of partial structures other than soluble proteins can be introduced into the same target region of a plurality of monomeric proteins.

In preferred embodiments, the soluble protein may be an antibody comprising multiple heavy chains. Definitions, examples and preferred examples of antibodies are given above. In the present invention, the number of partial structures other than antibodies possessed by antibodies is 1 or 2 (preferably 2) for IgG, IgE, and IgD, and 1 to 4 (preferably 4), and for IgM may be 1 to 8 (preferably 8).

5-5. Production Method The present invention provides a method for producing a soluble protein having a functional substance or a salt thereof.
(D) a method of using as raw materials a complex or a salt thereof having an affinity substance for a soluble protein, a cleavable moiety, a functional substance and a soluble protein (see reaction (3) in FIG. 2); and (E) a method using a soluble protein having a bioorthogonal functional group or a salt thereof as a starting material (see reaction (5) in FIG. 2).

(D) A method of using a conjugate having an affinity substance for a soluble protein, a cleavable moiety, a functional substance and a soluble protein or a salt thereof as a raw material includes:
(D1) cleaving the cleavable portion of a complex having an affinity substance for a soluble protein, a cleavable portion, a functional substance and a soluble protein or a salt thereof to produce a soluble protein having a functional substance or a salt thereof .

A conjugate comprising an affinity agent for a soluble protein, a cleaving moiety, a functional agent and a soluble protein is represented by formula (III), preferably formula (III'), more preferably formula (III''). A soluble protein having a functional substance is of formula (V), preferably of formula (V1), (V2) or (V3), more preferably of formula (V1'), (V2') or (V3') expressed.

A complex having an affinity substance for a soluble protein, a cleavable portion, a functional substance and a soluble protein or a salt thereof can be cleaved because it has a cleavable portion that can be cleaved by the above-described cleavage treatment. . Such a cleavage reaction can be carried out in the manner described in "4-6. Production method" above.

Confirmation of the production of a soluble protein having a functional substance or a salt thereof depends on the specific starting material and the molecular weight of the product, but for example, electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, chromatography, reversed-phase column chromatography, HPLC), or mass spectrometry, preferably mass spectrometry. Confirmation of regioselectivity, confirmation of the number of partial structures other than the soluble protein, and purification can be carried out as appropriate by the method described in "2-6. Production method" above.

The present invention also provides a method for producing a soluble protein having a functional substance or a salt thereof, comprising:
(D2) A conjugate having an affinity substance for a soluble protein, a cleavable portion, or a salt thereof containing a soluble protein affinity substance and a cleavable portion, or a salt thereof, with a functional substance to obtain a soluble protein-affinity substance, a cleavable portion, a functional substance, and a soluble protein and (D3) cleaving the cleavable portion of a complex having an affinity for a soluble protein, a cleavable moiety, a functional agent and a soluble protein or a salt thereof to yield a functional agent. to produce a soluble protein or a salt thereof having

The above step (D2) can be performed in the same manner as the above step (B1) described in "3-7. Manufacturing method". The step (D3) can be performed in the same manner as the step (D1). The above steps (D2) and (D3) can be performed separately. Alternatively, steps (D2) and (D3) above can be performed simultaneously, depending on factors such as combination with cleavable moieties, functional agents and bioorthogonal functional groups.

The present invention further provides a method for producing a soluble protein having a functional substance or a salt thereof, comprising:
(D4) reacting a compound or a salt thereof comprising an affinity substance for a soluble protein, a cleavable moiety and a reactive group with the soluble protein to produce an affinity substance for the soluble protein and a soluble protein comprising the cleavable moiety or a salt thereof; to generate;
(D5) A conjugate having an affinity substance for a soluble protein, a cleavable portion, or a salt thereof containing a soluble protein affinity substance, a cleavable portion, a functional substance and a soluble protein by reacting with a functional substance and (D6) cleaving the cleavable portion of a complex having an affinity for a soluble protein, a cleavable moiety, a functional agent and a soluble protein or a salt thereof to yield a functional agent. to produce a soluble protein or a salt thereof having

The above step (D4) can be performed in the same manner as the above step (A1) described in "2-6. Production method" above. The steps (D5) and (D6) above can be performed in the same manner as the steps (D2) and (D3) above. The above step (D5) can be performed in the same manner as the above step (B1) described in the above "3-7. Manufacturing method". The step (D6) can be performed in the same manner as the step (D1). The above steps (D4) to (D6) can be performed separately. Alternatively, at least some of the steps (D4) to (D6) above can be performed simultaneously, depending on factors such as the combination with reactive groups, cleavable moieties, functional agents and bioorthogonal functional groups. .

(E) A method using a soluble protein having a bioorthogonal functional group or a salt thereof as a raw material includes:
(E1) Reacting a soluble protein having a bioorthogonal functional group or a salt thereof with a functional substance to produce a soluble protein having a functional substance or a salt thereof.

Soluble proteins with bioorthogonal functional groups are represented by formula (IV), preferably formula (IV'). A soluble protein having a functional substance is of formula (V), preferably of formula (V1), (V2) or (V3), more preferably of formula (V1'), (V2') or (V3') expressed.

A soluble protein having a bioorthogonal functional group or a salt thereof can react with a functional agent because it has a bioorthogonal functional group. Such a reaction can be carried out in the manner described in "3-7. Production method" above.

Confirmation of production of a soluble protein having a functional substance or a salt thereof can be performed by the methods described above. Confirmation of regioselectivity, confirmation of the number of partial structures other than the soluble protein, and purification can be carried out as appropriate by the method described in "2-6. Production method" above.

The present invention also provides a method for producing a soluble protein having a functional substance or a salt thereof, comprising:
(E2) Cleavage of a soluble protein or salt thereof comprising an affinity agent for a soluble protein and a cleavable moiety to produce a soluble protein or salt thereof having a bioorthogonal functional group; and (E3 ) reacting a soluble protein having a bioorthogonal functional group or a salt thereof with a functional agent to produce a soluble protein having a functional agent or a salt thereof;

The above step (E2) can be performed in the same manner as the above step (C1) described in "4-6. Production method" above. The step (E3) can be performed in the same manner as the step (E1). The above steps (E2) and (E3) can be performed separately. Alternatively, steps (E2) and (E3) above can be performed simultaneously, depending on factors such as combination with cleavable moieties, functional agents and bioorthogonal functional groups.

The present invention further provides a method for producing a soluble protein having a functional substance or a salt thereof, comprising:
(E4) reacting a compound or a salt thereof comprising an affinity substance for a soluble protein, a cleavable moiety and a reactive group with a soluble protein to produce an affinity substance for a soluble protein and a soluble protein comprising a cleavable moiety or a salt thereof; to generate;
(E5) Cleavage of a soluble protein or salt thereof comprising an affinity agent for a soluble protein and a cleavable moiety to produce a soluble protein or salt thereof having a bioorthogonal functional group; and (E6 ) reacting a soluble protein having a bioorthogonal functional group or a salt thereof with a functional agent to produce a soluble protein having a functional agent or a salt thereof;

The above step (E4) can be performed in the same manner as the above step (A1) described in the above “2-6. Manufacturing method”. The steps (E5) and (E6) above can be performed in the same manner as the steps (E2) and (E3) above. The above steps (E4) to (E6) can be performed separately. Alternatively, at least some of the steps (E4)-(E6) above can be performed simultaneously, depending on factors such as the combination with reactive groups, cleavable moieties, functional agents and bioorthogonal functional groups. .

6. Specific Soluble Proteins Regiospecifically Having Bioorthogonal Functional Groups or Salts Thereof, and Production Methods Thereof The present invention also provides soluble proteins or salts thereof regioselectively having bioorthogonal functional groups. The soluble protein or a salt thereof of the present invention regioselectively having a bioorthogonal functional group has one or more specific amino acid residues in the target region consisting of 1 to 50 consecutive amino acid residues. and 5 or more of the specific amino acid residues in a non-target region other than the target region, and the bioorthogonal functional group is included in the target region via a linker that does not contain a peptide portion. It is a soluble protein or a salt thereof regioselectively having a bioorthogonal functional group, which is bound to one or more specific amino acid residues with regioselectivity of 30% or more.

In certain embodiments, a soluble protein of the invention or a salt thereof regioselectively bearing a bioorthogonal functional group has the following formula (IV):
L1-B-R'-T (IV)
[In the formula,
L1 is (i′) a monovalent group containing a bioorthogonal functional group or (ii′) a monovalent group not containing a bioorthogonal functional group;
B is (a) a divalent group that includes a bioorthogonal functional group or (b) a divalent group that does not include a bioorthogonal functional group;
R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein,
The soluble protein contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and the specific amino acid residue in a non-target region other than the target region 5 or more. ] and the structural unit represented by L1-BR' has a regioselectivity of 30% or more with respect to one or more specific amino acid residues contained in the target region of the soluble protein It may be a soluble protein or a salt thereof that regioselectively has a bioorthogonal functional group attached.

The present invention also provides a method for producing a soluble protein or a salt thereof regioselectively having a bioorthogonal functional group.

With conventional methods, it was not possible to regioselectively produce a soluble protein or a salt thereof having a bioorthogonal functional group as described above. Soluble proteins or salts thereof regioselectively possessing bioorthogonal functional groups can be produced. requirements such as bioorthogonal functional groups, regioselectivity, soluble proteins, targeting regions, linkers, and (i′) and (ii′) in L1, (a) and (b) in B, R′, and T, Definitions, examples and preferred examples of the technical elements and some of those requirements are the same as those described above. The details of the steps of the production method are also described in "4. Production of a soluble protein having a bioorthogonal functional group or a salt thereof, except that regioselectivity and technical elements related thereto (e.g., target region) are essential requirements. method”.

7. Soluble Protein Regioselectively Having a Functional Substance or its Salt, and Production Method Thereof The present invention also provides a soluble protein having a functional substance regioselectively or a salt thereof. The soluble protein or salt thereof of the present invention having a functional substance regioselectively contains one or more specific amino acid residues in the target region consisting of 1 to 50 consecutive amino acid residues, At least one of the functional substances contained in the target region via a linker that does not contain a peptide portion, and contains 5 or more of the specific amino acid residues in a non-target region other than the target region. is a soluble protein or a salt thereof that regioselectively has a functional substance that binds to a specific amino acid residue of , with a regioselectivity of 30% or more.

In certain embodiments, the soluble protein or salt thereof of the present invention regioselectively possessing a functional agent has the following formula (V):
F-(L1-B)'-R'-T (V)
[In the formula,
L1 is (i′) a monovalent group containing a bioorthogonal functional group or (ii′) a monovalent group not containing a bioorthogonal functional group;
B is (a) a divalent group that includes a bioorthogonal functional group or (b) a divalent group that does not include a bioorthogonal functional group;
The structural unit represented by (L1-B)' includes a moiety generated by a reaction between a functional substance and either one or both of the bioorthogonal functional groups in (i') and (a). is a divalent structural unit,
F is a functional substance,
R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein,
The soluble protein contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and the specific amino acid residue in a non-target region other than the target region 5 or more. ] and the structural unit represented by F-(L1-B)'-R' accounts for 30% or more of one or more specific amino acid residues contained in the target region of the soluble protein It may be a soluble protein or a salt thereof that regioselectively has a functional substance that is bound regioselectively.

The present invention also provides a method for producing a soluble protein having a functional substance regioselectively or a salt thereof.

With conventional methods, it was not possible to produce soluble proteins or salts thereof regioselectively having functional substances as described above. A soluble protein or a salt thereof regioselectively possessing a soluble substance can be produced. Requirements for functional substances, regioselectivity, soluble proteins, target regions, linkers, etc., and (i′) and (ii′) in L1, (a) and (b) in B, (L1
-B) ', F, R', and T, and definitions, examples and preferred examples of some of their requirements are the same as those described above. The details of the steps of the production method are also described in "5. Method for producing a soluble protein having a functional substance or a salt thereof", except that regioselectivity and related technical elements (e.g., target region) are essential requirements. is the same as described in .

8. Salts In the present invention, salts include, for example, salts with inorganic acids, salts with organic acids, salts with inorganic bases, salts with organic bases, and salts with amino acids. Salts with inorganic acids include, for example, salts with hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, and nitric acid. Examples of salts with organic acids include formic acid, acetic acid, trifluoroacetic acid, lactic acid, tartaric acid, fumaric acid, oxalic acid, maleic acid, citric acid, succinic acid, malic acid, benzenesulfonic acid, and p-toluenesulfonic acid. salt of Salts with inorganic bases include, for example, salts with alkali metals (eg, sodium, potassium), alkaline earth metals (eg, calcium, magnesium), and other metals such as zinc, aluminum, and ammonium. . Salts with organic bases include, for example, salts with trimethylamine, triethylamine, propylenediamine, ethylenediamine, pyridine, ethanolamine, monoalkylethanolamine, dialkylethanolamine, diethanolamine, and triethanolamine. Examples of salts with amino acids include salts with basic amino acids (eg, arginine, histidine, lysine, ornithine) and acidic amino acids (eg, aspartic acid, glutamic acid). The salt is preferably a salt with an inorganic acid (eg hydrogen chloride) or an organic acid (eg trifluoroacetic acid).

9. Uses Compounds of the present invention or salts thereof having affinity for soluble proteins, cleavable moieties and reactive groups are useful, for example, for regioselective modification of soluble proteins. Accordingly, the present invention provides regioselective modification reagents for soluble proteins, including compounds having an affinity for soluble proteins, cleavable moieties and reactive groups or salts thereof.

The regioselective modification reagent of the present invention may be provided in the form of a composition that further contains other components. Such other ingredients include, for example, solutions, stabilizers (eg, antioxidants, preservatives). As the solution, an aqueous solution is preferred. Aqueous solutions include, for example, water (eg, distilled water, sterilized distilled water, purified water, physiological saline), buffers (eg, phosphoric acid aqueous solution, Tris-hydrochloric acid buffer, carbonate-bicarbonate buffer, boric acid aqueous solution). , glycine-sodium hydroxide buffer, citrate buffer), but buffers are preferred. The pH of the solution is, for example, 5.0-9.0, preferably 5.5-8.5. The regioselective modification reagents of the invention can be provided in liquid or powder form (eg, lyophilized powder).

The affinity substance for soluble protein, and the soluble protein or salt thereof of the present invention having (regioselectively) a cleavable moiety, the affinity substance for soluble protein, the cleavable moiety, the functional substance and the soluble protein (regioselectively The conjugate of the present invention or a salt thereof having (regioselectively) a soluble protein of the present invention having a bioorthogonal functional group (regioselectively) or a salt thereof having, for example, a soluble It is useful as an intermediate for the preparation of proteins or salts thereof.

The soluble protein or salt thereof of the present invention having a functional substance (regioselectively) is useful, for example, as a drug or reagent (eg, diagnostic agent, research reagent). In particular, when the soluble protein is an antibody, the antibody or salt thereof of the present invention having a functional substance (regioselectively) is suitable for these uses.

In particular, the antibody or salt thereof of the present invention having a functional substance (regioselectively) is useful as a pharmaceutical. As described above, it has been reported that changes in the binding number and binding position of the drug in the antibody-drug conjugate (ADC) change the pharmacokinetics, drug release rate, and effect. For these reasons, next-generation ADCs are required to control the number and positions of drugs to be conjugated. It is believed that a constant number and position solves the problem of expected efficiency, variations in conjugation drugs, lot differences, so-called regulation. Antibodies or salts thereof of the present invention having functional substances can solve such regulation problems. Accordingly, the antibody or salt thereof of the present invention having a functional substance may be provided in the form of a pharmaceutical composition. Such pharmaceutical compositions may contain a pharmaceutically acceptable carrier in addition to the soluble protein having a functional substance or a salt thereof. Pharmaceutically acceptable carriers include, for example, excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate, cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone. , gelatin, gum arabic, polyethylene glycol, sucrose, starch and other binders, starch, carboxymethylcellulose, hydroxypropyl starch, sodium hydrogen carbonate, calcium phosphate, calcium citrate and other disintegrants, magnesium stearate, aerosil, talc, lauryl Lubricants such as sodium sulfate, citric acid, menthol, glycyrrhizin ammonium salt, glycine, fragrances such as orange powder, preservatives such as sodium benzoate, sodium hydrogen sulfite, methylparaben, propylparaben, citric acid, sodium citrate, acetic acid Stabilizers such as methylcellulose, polyvinylpyrrolidone, aluminum stearate, suspending agents such as surfactants, dispersing agents such as surfactants, diluents such as water, physiological saline, orange juice, cacao butter, polyethylene glycol, white kerosene, etc. Examples include, but are not limited to, base waxes. Antibodies or salts thereof of the present invention having functional agents may also have optional modifications (eg, PEGylation) that provide stability.

Formulations suitable for oral administration include liquid formulations in which an effective amount of the ligand is dissolved in a diluent such as water, saline or orange juice, capsules, sachets or tablets containing an effective amount of the ligand as a solid or granules. Examples include tablets, suspensions in which an effective amount of the active ingredient is suspended in a suitable dispersion medium, and emulsions in which a solution in which an effective amount of the active ingredient is dissolved is dispersed and emulsified in a suitable dispersion medium.

The pharmaceutical composition is suitable for parenteral administration (eg, intravenous injection, subcutaneous injection, intramuscular injection, local injection, intraperitoneal administration). Pharmaceutical compositions suitable for such parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions containing antioxidants, buffers, bacteriostatic agents, tonicity agents. etc. may be included. Also included are aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives and the like.

The dose of the pharmaceutical composition varies depending on the type and activity of the active ingredient, severity of disease, animal species to be administered, drug acceptability, body weight, age, etc. of the subject, but can be set as appropriate.

EXAMPLES Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[Example 1: Synthesis of IgG1 Fc-binding peptide]
(1) Synthesis of IgG1 Fc-binding peptide (1-1) Synthesis of substance with affinity for soluble protein (peptide of Ac-RGNCAYHKGQLVWCTYH-NH ₂ (SEQ ID NO: 39)) Ac-RGNCAYHKGQLVWCTY which is an affinity substance for soluble protein
A peptide of H—NH ₂ (SEQ ID NO:39) was synthesized by Fmoc solid-phase synthesis. Syrowave manufactured by Biotage was used as a peptide synthesizer. All the reagents used were those manufactured by Watanabe Chemical Industry. Resin was Fmoc-NH-SAL-PEG Resin, HL, arginine (R), cysteine (C), and histidine (H) were double-coupled. Cutting from Resin was performed in a solution of trifluoroacetic acid:water:triisopropylsilane:ethanedithiol=94:2.5:1.0:2.5 under stirring conditions for 3 hours. After cutting out, the resin was removed by filtration, and the trifluoroacetic acid was removed. Diethyl ether was added to the resulting crystals for ether precipitation, and the resulting white crystals were collected by filtration. This was dissolved in an aqueous 0.1% trifluoroacetic acid solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. Elution was performed with the mixed solution, and each fraction was confirmed by LC-MS. The fraction containing the product was collected, concentrated under reduced pressure to remove only acetonitrile, and then lyophilized.

MS (ESI) m/z: z=3 693 [M+3H] ^<3+> , z=4 520 [M+4H] ^<4+>

上記アミノ酸配列は、配列番号３９である。 (1-2) Formation of an intramolecular disulfide bond at Cys at positions 4 and 14 in the peptide of Ac-RGNCAYHKGQLVWCTYH-NH ₂ (SEQ ID NO: 39)

The amino acid sequence is SEQ ID NO:39.

After dissolving the peptide synthesized in (1-1) in DMSO to 100 mM, 2 equivalents of 2M NH ₃ -MeOH and 1 equivalent of aqueous hydrogen peroxide were added and stirred at room temperature for 12 hours. A 0.1% trifluoroacetic acid aqueous solution was added to the reaction solution to stop the reaction, elution was performed by reverse phase preparative chromatography, and each fraction was confirmed by LC-MS. The fraction containing the product was collected, concentrated under reduced pressure to remove only acetonitrile, and then lyophilized. Yields were above 90% on a scale of 5 mg to 100 mg.

MS (ESI) m/z: z = 3 692 [M+3H] ³⁺ , z = 4 519 [M+4H] ⁴⁺

[Example 2: Synthesis of cleavable linker and ligation with IgG1 Fc-binding peptide]
(2) Synthesis of cleavable linker and ligation with peptide (2-1) Synthesis of thioester linker and ligation with peptide (2-1-1) Synthesis of thioester linker

Succinic anhydride (0.2 g, 2 mmol) and N,N'-dimethylaminopyridine (12.2 mg, 0.1 mmol) were dissolved in a solvent of acetonitrile:pyridine=9:1 and purged with argon gas. tert-butyl-3-sulfanylpropanoate (0.3 g,
2.2 mmol) was added and stirred at room temperature for 16 hours. The reaction solution was diluted with ethyl acetate, washed with 0.1M hydrochloric acid aqueous solution, water and brine, added with anhydrous magnesium sulfate, and allowed to stand for 5 minutes. Magnesium sulfate was removed by filtration and concentration under reduced pressure gave 4-(3-tert-butoxy-3-oxo-propyl)sulfanyl-4-oxo-butanoic acid (0.47 g, 1.8 mmol). .

¹ H NMR (400 MHz, Chloroform-d) δ 3.12 (t, J=7.0 Hz, 2H), 2.91 (t, J=6.9 Hz, 2H), 2.72 (t, J=6 .9 Hz, 2 H), 2.55 (t, J=7.0 Hz, 2 H), 1.46 (s, 9 H).
MS (ESI) m/z: 263 [M+H] ⁺

5 mL of ethyl acetate was added to dissolve 4-(3-tert-butoxy-3-oxo-propyl)sulfanyl-4-oxo-butanoic acid (0.47 g, 1.8 mmol). After that, N,N'-dicyclohexylcarbodiimide (0.39 g, 1.9 mmol) and N-hydroxysuccinimide (0.22 g, 1.9 mmol) were added and stirred for 1 hour. The resulting white crystals are filtered and the mother liquor is concentrated under reduced pressure to give (2,5-dioxopyrrolidin-1-yl).
4-(3-tert-butoxy-3-oxo-propyl)sulfanyl-4-oxo-butanoate (0.61 g, 1.7 mmol) was obtained.

MS (ESI) m/z: 360 [M+H] ⁺

上記アミノ酸配列は、配列番号３９である。 (2-1-2) Linking of thioester linker and peptide

The amino acid sequence is SEQ ID NO:39.

The peptide (42 mg, 20.3 μmol) of Ac-RGNCAYHKGQLVWCTYH-NH ₂ (SEQ ID NO: 39) synthesized in Example 1-2, provided that the two cysteines at the 4th and 14th positions each form an intramolecular disulfide bond. (2,5-dioxopyrrolidin-1-yl) 4-(3-tert-butoxy-3-oxo-propyl)sulfanyl-4-oxo -Butanoate (0.61 g, 1.7 mmol) was added into a solution in 1 mL of acetonitrile. After stirring at room temperature for 12 hours, 0.1% trifluoroacetic acid aqueous solution was added and elution was performed by reverse phase preparative chromatography. Fractions containing the product were collected and concentrated under reduced pressure to remove only acetonitrile, followed by freeze-drying to obtain the peptide linker-linked-tBu form (41.7 mg, 18 μmol).

MS (ESI) m/z: z=3 773 [M+3H] ^<3+> , z=4 580 [M+4H] ^<4+>

上記アミノ酸配列は、配列番号３９である。

The amino acid sequence is SEQ ID NO:39.

The above peptide linker conjugate (41.7 mg, 18 μmol) was dissolved in 1.0 mL of dichloromethane, 1.0 mL of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 1 hour. After concentrating under reduced pressure, 0.1% trifluoroacetic acid aqueous solution was added and elution was performed by reverse phase preparative chromatography. Fractions containing the product were collected and concentrated under reduced pressure to remove only acetonitrile, followed by freeze-drying to obtain the peptide linker conjugate-carboxylic acid form (40.7 mg, 18 μmol).

MS (ESI) m/z: z=3 755 [M+3H] ^<3+> , z=4 566 [M+4H] ^<4+>

上記アミノ酸配列は、配列番号３９である。

The amino acid sequence is SEQ ID NO:39.

The above peptide linker conjugate-carboxylic acid form (40.7 mg, 18 μmol) was dissolved in 1 mL of N,N'-dimethylformamide, and N,N'-dicyclohexylcarbodiimide (37.1 mg, 0.18 mmol) and N-hydroxysuccinimide were added. (20.7 mg, 0.18 mmol) was added and stirred for 1 hour. The resulting white crystals were filtered, and 0.1% trifluoroacetic acid aqueous solution was added to the mother liquor and eluted by reverse phase preparative chromatography. Fractions containing the product were collected and concentrated under reduced pressure to remove only acetonitrile, followed by freeze-drying to obtain the peptide thioester linker conjugate-NHS activated product (5.2 mg, 2.24 μmol). .

MS (ESI) m/z: z=3 787 [M+3H] ^<3+> , z=4 590 [M+4H] ^<4+>

上記アミノ酸配列は、配列番号３９である。 (2-2) Linking disulfide linker and peptide

The amino acid sequence is SEQ ID NO:39.

The peptide (10 mg, 4.8 μmol) of Ac-RGNCAYHKGQLVWCTYH-NH ₂ (SEQ ID NO: 39) synthesized in Example 1-2, provided that the two cysteines at the 4th and 14th positions each form an intramolecular disulfide bond. ) was dissolved in 1 mL of N,N'-dimethylformamide, and added to a solution of di(N-succinimidyl) 3,3'-dithiodipropionate (0.2 g, 0.48 mmol) dissolved in 1 mL. and stirred for 12 hours. A 0.5% trifluoroacetic acid aqueous solution was added, and elution was performed by reverse phase preparative chromatography. Fractions containing the product were collected and concentrated under reduced pressure to remove only acetonitrile, followed by freeze-drying to obtain the peptide disulfide linker conjugate-NHS activated product (8.6 mg, 3.63 μmol). .

MS (ESI) m/z: z=3 789 [M+3H] ^<3+> , z=4 592 [M+4H] ^<4+>

(2-3) Synthesis of acetal linker and ligation of peptide (2-3-1) Synthesis of acetal linker

Pentaerythritol (1.98 g, 14.3 mmol), methyl dimethoxypropionate (6.0 g, 31.7 mmol) and p-toluenesulfonic acid monohydrate (33.0 mg, 0.143 mmol) were mixed, and 130 °C for 16 hours. After 16 hours, the temperature was returned to room temperature, the precipitated crystals were collected by filtration, and methyl 2-[9-(2-methoxy-2-oxo-ethyl)-2,4,8,10-tetraoxospiro[5.5]undecane was obtained. -3-yl]acetate (3.04 g, 10 mmol) was obtained.

MS (ESI) m/z: 305 [M+H] ⁺

Methyl 2-[9-(2-methoxy-2-oxo-ethyl)-2,4,8,10-tetraoxospiro[5.5]undecane-3-yl]acetate (50mg, 0.164m
mol) was dissolved in 1 mL of methanol and dissolved in 1 M NaOH aq. 0.49 mL was added, and the temperature was raised to room temperature, followed by stirring for 2 hours. After neutralization by adding a cation exchange resin, the resin was removed by filtration and 2-[9-(2-methoxy-2-oxo-ethyl)-2,4,8,10-tetraoxospiro[5 .5]undecane-3-yl]acetic acid (32 mg, 0.116 mmol) was obtained.

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 4.85 (t, J = 5.4 Hz, 2H), 4.26 (dd, J = 11.3, 2.4 Hz, 2H), 3.67- 3.61 (m, 2H), 3.60 (s, 8H), 3.42 (d, J=11.6Hz, 2H), 2.69-2.56 (m, 4H).
MS (ESI) m/z: 277 [M+H] ⁺

2-[9-(2-Methoxy-2-oxo-ethyl)-2,4,8,10-tetraoxospiro[5.5]undecane-3-yl]acetic acid (32 mg, 0.116 mmol) was added to ethyl acetate. The solution was dissolved in 1 mL, N,N'-dicyclohexylcarbodiimide (37.1 mg, 0.18 mmol) and N-hydroxysuccinimide (20.7 mg, 0.18 mmol) were added and stirred for 1 hour. The resulting white crystals were filtered, the mother liquor was concentrated under reduced pressure, water was added, and neutral acetonitrile and water were used as solvents for elution by reverse phase preparative chromatography. The fraction containing the product is collected and concentrated under reduced pressure to remove only acetonitrile, followed by freeze-drying, (2,5-dioxopyrrolidin-1-yl) 2-[9-(2-methoxy- 2-Oxo-ethyl)-2,4,8,10-tetraoxospiro[5.5]undecane-3-yl]acetate (13 mg, 27.6 μmol) was obtained.

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 4.79 (t, J = 5.3 Hz, 2H), 4.24 (dd, J = 11.2, 2.3 Hz, 2H), 3.60- 3.48 (m, 4H), 3.34 (d, J=11.5Hz, 2H), 2.33 (d, J=5.3Hz, 4H).
MS (ESI) m/z: 471 [M+H] ⁺

上記アミノ酸配列は、配列番号３９である。 (2-3-2) Linking of acetal linker and peptide

The amino acid sequence is SEQ ID NO:39.

The peptide (10 mg, 4.8 μmol) of Ac-RGNCAYHKGQLVWCTYH-NH ₂ (SEQ ID NO: 39) synthesized in Example 1-2, provided that the two cysteines at the 4th and 14th positions each form an intramolecular disulfide bond. ) was dissolved in 1 mL of N,N′-dimethylformamide, and (2,5-dioxopyrrolidin-1-yl) 2-[9-(2-methoxy-2-oxo-ethyl)-2,4 , 8,10-tetraoxospiro[5.5]undecane-3-yl]acetate (13 mg, 27.6 μmol) dissolved in 1 mL was added and stirred at room temperature for 12 hours. Water was added and neutral acetonitrile and water were used as solvents for elution by reverse phase preparative chromatography. Fractions containing the product were collected and concentrated under reduced pressure to remove only acetonitrile, followed by freeze-drying to obtain the peptide acetal linker conjugate-NHS activated product (8.6 mg, 3.63 μmol). .

MS (ESI) m/z: z=3 810 [M+3H] ^<3+> , z=4 608 [M+4H] ^<4+>

[Example 3: Specific modification of IgG1 Fc by IgG1 Fc-binding peptide reagent and analysis by MALDI-TOFMS]
(3-1) Specific modification of IgG1 Fc with a thioester linker-binding peptide reagent and analysis by MALDI-TOFMS IgG1 Fc is manufactured by R & D Co. IgG1 Fc, Human, Recombinant, Carrier-free (product number: 110-HG-100 ) was used. The peptide thioester linker conjugate-NHS-activated product synthesized in (2-1) of Example 2 was dissolved in N,N'-dimethylformamide to a concentration of 0.4 mM. 15 μg of IgG1 Fc was dissolved in 15 μL of 0.1 M sodium acetate buffer (pH 4.7), 7 μL of 0.4 mM peptide reagent (5 equivalents to IgG1 Fc) was added, and the mixture was stirred at room temperature for 1 hour. The solvent of the reaction solution was replaced with 9.57 mM PBS buffer (pH 7.4) using Amicon 10K to terminate the reaction. When the mass was measured by MALDI-TOFMS, a peak was observed at m/z 28270 for the raw material IgG1 Fc, and a peak was observed at m/z 30532 for the product into which one binding peptide was introduced.

(3-2) Specific modification of IgG1 Fc with a disulfide linker-binding peptide reagent and analysis by MALDI-TOFMS IgG1 Fc is manufactured by R & D Co. IgG1 Fc, Human, Recombinant, Carrier-free (product number: 110-HG-100 ) was used. The peptide disulfide linker conjugate-NHS-activated product synthesized in (2-2) of Example 2 was dissolved in N,N'-dimethylformamide to a concentration of 0.4 mM. 20 μg of IgG1 Fc was dissolved in 40 μL of 0.1 M sodium acetate buffer (pH 4.7), 10.7 μL of 0.4 mM peptide reagent (6 equivalents to IgG1 Fc) was added, and the mixture was stirred at room temperature for 1 hour. The solvent of the reaction solution was replaced with 9.57 mM PBS buffer (pH 7.4) using Amicon 10K to terminate the reaction. When the mass was measured by MALDI-TOFMS, a peak was observed at m/z 28183 for the raw material IgG1 Fc, and a peak was observed at m/z 30418 for the product into which one binding peptide was introduced.

(3-3) Specific modification of IgG1 Fc with an acetal linker-binding peptide reagent and analysis by MALDI-TOFMS IgG1 Fc is manufactured by R & D Co. IgG1 Fc, Human, Recombinant, Carrier-free (product number: 110-HG-100 ) was used. The peptide acetal linker conjugate-NHS-activated product synthesized in (2-3) of Example 2 was dissolved in N,N'-dimethylformamide to a concentration of 0.4 mM. 20 μg of IgG1 Fc was dissolved in 40 μL of 0.1 M sodium acetate buffer (pH 4.7), 10.7 μL of 0.4 mM peptide reagent (6 equivalents to IgG1 Fc) was added, and the mixture was stirred at room temperature for 1 hour. The solvent of the reaction solution was replaced with 9.57 mM PBS buffer (pH 7.4) using Amicon 10K to terminate the reaction. When the mass was measured by MALDI-TOFMS, a peak was observed at m/z 28183 for the raw material IgG1 Fc, and a peak was observed at m/z 30541 for the product into which one binding peptide had been introduced.

[Example 4: Linker cleavage of IgG1 Fc-peptide complex and analysis by MALDI-TOFMS]
(4-1) Cleavage of thioester linker and analysis by MALDI-TOFMS 0.2 M PBS buffer (pH 7.0), 40 μL of 25 mM cysteine solution, IgG1 Fc-peptide thioester linker synthesized in (3-1) of Example 3 The complex was dissolved and stirred at room temperature for 16 hours. The solvent of the reaction solution was replaced with 9.57 mM PBS buffer (pH 7.0) using Amicon 10K to terminate the reaction. When the mass was measured by MALDI-TOFMS, the m/z
A peak was observed at 30541 and a peak was observed at m/z 28397 after the reaction.

(4-2) Cleavage of thioester linker and analysis by MALDI-TOFMS Dissolve the IgG1 Fc-peptide disulfide linker complex synthesized in (3-2) of Example 3 in 40 μL of 0.2 M PBS buffer (pH 7.0). Then, 12.5 μL of 10 mM D,L-dithiothreitol (10 equivalents to Fc) was added and stirred at room temperature for 16 hours. The solvent of the reaction solution was replaced with 9.57 mM PBS buffer (pH 7.0) using Amicon 10K to terminate the reaction. When the mass was measured by MALDI-TOFMS, a peak was observed at m/z 30418 for the complex into which one starting binding peptide had been introduced, and a peak was observed at m/z 28183 after the reaction.

(4-3) Cleavage of acetal linker and analysis by MALDI-TOFMS The IgG1 Fc-peptide acetal linker complex synthesized in (3-3) of Example 3 was added to an arbitrary buffer (pH 3.0 to pH 6.0). Upon dissolution and stirring at room temperature, the acetal structure decomposes leaving the aldehyde on the IgG1 Fc. The mass of this can be measured by MALDI-TOFMS.

[Example 5: Specific modification of anti-HER2 IgG antibody trastuzumab and anti-CD20 antibody rituximab and analysis by MALDI-TOFMS and HIC-HPLC]
(5-1) Specific modification of anti-HER2 IgG antibody trastuzumab and analysis by MALDI-TOFMS The peptide disulfide linker conjugate-NHS-activated product synthesized in (2-2) of Example 2 is N,N'-dimethylformamide to 0.4 mM. Dissolve 500 μg of anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical Co., Ltd.) in 200 μL of 50 mM sodium acetate buffer (pH 5.5), add 50.7 μL of 0.4 mM peptide reagent (6 equivalents to the antibody), and warm to room temperature. and stirred for 1 hour. The solvent of the reaction solution was replaced with 9.57 mM PBS buffer (pH 7.4) using Amicon 10K to terminate the reaction. When the mass was measured by MALDI-TOFMS, a peak was observed at m/z 147569 for the raw material trastuzumab, and a peak was observed at m/z 151848 for the product into which two binding peptides were introduced.

(5-2) HIC-HPLC Analysis of Specific Modification of Anti-HER2 IgG Antibody Trastuzumab The antibody/peptide complex produced in (5-1) and the starting antibody were analyzed by HIC. A Butyl-NPR (TOSOH) 4.6×350 mm 2.5 μm column was used. A_Buffer: 0.1 M PiNa, 2.3 M (NH ₄ ) ₂ SO ₄ , pH 7.0, B_Buffer: 0.1 M PiNa, pH 7.0, flow rate 1.0 ml/min, gradient B 0→100%, Detection was performed for 12 minutes (data collection: 20 minutes), the column temperature was 25° C., the thermostat temperature was RT, and the detector was UV 225 nm (Ch1) and 280 nm (Ch2).

The samples were reacted under the following conditions.
a: Trastuzumab + peptide reagent 12 equivalents (solvent replacement with Amicon 10K after reaction);
b: Trastuzumab + 12 equivalents of peptide reagent;
c: Trastuzumab + 6 equivalents of peptide reagent;
d: trastuzumab raw material;
e: peptide reagent only;
f: DMF only.

Retension Time 9.417 minutes is the trastuzumab raw material, 10.943 minutes and 13.063 minutes are peaks derived from peptide reagents, and 0.44 minutes is DMF (Figs. 3 and 4). When trastuzumab was reacted with 6 equivalents of the peptide reagent, the peak at 9.417 minutes disappeared and peaks at 9.99 minutes and 10.237 minutes appeared (Figs. 3 and 4). When 12 equivalents of the peptide reagent were added, the peak at 9.99 minutes disappeared, suggesting that the peak at 9.99 minutes was a compound in which one peptide had been introduced into trastuzumab (Figs. 3 and 4). . When a was analyzed by MALDI-TOFMS, as shown in (5-1), two peaks were observed in which two peptide reagents were introduced into trastuzumab showing MS peaks of m/z 151848 (Fig. 3, 4).

(5-3) Specific Modification of Anti-CD20 Antibody Rituximab and Analysis by MALDI-TOFMS It was dissolved to 0.4 mM. 500 µg of anti-CD20 antibody rituximab was dissolved in 200 µL of 50 mM sodium acetate buffer (pH 5.5), 50.7 µL of 0.4 mM peptide reagent (6 equivalents to the antibody) was added, and the mixture was stirred at room temperature for 1 hour. The solvent of the reaction solution was replaced with 9.57 mM PBS buffer (pH 7.4) using Amicon 10K to terminate the reaction. When the mass was measured by MALDI-TOFMS, a peak was observed at m/z 144002 for the raw material rituximab, and a peak was observed at m/z 148169 for the product into which two binding peptides were introduced.

(5-4) HIC-HPLC Analysis of Specific Modification of Anti-CD20 Antibody Rituximab The antibody/peptide complex produced in (5-3) and the starting antibody were analyzed by HIC. A Butyl-NPR (TOSOH) 4.6×350 mm 2.5 μm column was used. A_Buffer: 0.1 M PiNa, 2.3 M (NH ₄ ) ₂ SO ₄ , pH 7.0, B_Buffer: 0.1 M PiNa, pH 7.0, flow rate 1.0 ml/min, gradient B 0→100%, Detection was performed for 12 minutes (data collection: 20 minutes), the column temperature was 25° C., the thermostat temperature was RT, and the detector was UV 225 nm (Ch1) and 280 nm (Ch2).

Samples a to f were reacted under the following conditions.
a: Rituximab + peptide reagent 12 equivalents (solvent replacement with Amicon 10K after reaction);
b: Rituximab + 12 equivalents of peptide reagent;
c: Rituximab + 6 equivalents of peptide reagent;
d: Rituximab raw material;
e: peptide reagent only;
f: DMF only.

Retension Time 9.417 minutes is the Rituximab raw material, 10.94 minutes and 13.02 minutes are peaks derived from the peptide reagent, and 0.44 minutes is DMF (Figs. 5 and 6). When rituximab was reacted with 6 equivalents of the peptide reagent, the peak at 10.36 minutes disappeared and peaks at 10.763 minutes and 10.94 minutes appeared (Figs. 5 and 6). When 12 equivalents of the peptide reagent were added, the peak at 10.763 minutes disappeared, suggesting that the peak at 10.763 minutes was a compound in which one peptide was introduced into rituximab (Figs. 5 and 6). . When a was analyzed by MALDI-TOFMS, two peaks with m/z 148169 peptide reagent introduced into rituximab were observed as shown in (5-1) (FIGS. 5 and 6).

[Example 6: Cleavage and reoxidation of trastuzumab-peptide complexes and rituximab-peptide complexes, and analysis of their products by MALDI-TOFMS and RP-HPLC]
(6-1) Linker Cleavage and Reoxidation of Trastuzumab Peptide Complex, and Analysis of Their Products by MALDI-TOFMS First, 400 μg of the trastuzumab peptide complex synthesized in (5-1) of Example 5 was After dissolving in phosphate buffer (pH 8.0) to 72 μM, 10.0 μL of 20 mM D,L-dithiothreitol (60 equivalents to trastuzumab peptide complex) was added and stirred at room temperature for 16 hours. to cleave the disulfide bond in the linker.

Next, a reoxidation step was performed in order to rebond the disulfide bonds in the antibody that had been cleaved together with the disulfide bonds in the linker (Jagath R Junutula et al., NATURE BIOTECHNOLOGY, 26(8), 925-932 ( 2008)). Specifically, the reaction solution was solvent-exchanged with 9.57 mM PBS buffer (pH 7.0) using Amicon 10K to adjust the concentration of the trastuzumab-peptide complex to 18 µM, and then 6.4 µL of 10 mM dehydroascorbic acid (trastuzumab-peptide complex) was added. 20 equivalents relative to the peptide conjugate) and stirred for 3 hours to effect reoxidation. When the mass was measured by MALDI-TOFMS, a peak was observed at m/z 145329 where two thiopropionyl groups were introduced into the product.

(6-2) Analysis by RP-HPLC of Products Obtained by Linker Cleavage and Reoxidation of Trastuzumab-Peptide Complex The reaction in (6-1) was analyzed by RP-HPLC. The column used was TSK gel, Protein C4-300 (TOSOH) 4.6×150 mm 3.0 μm. A_Buffer: 0.1% TFA, B_Buffer: 0.1% TFA, 80% ACN, flow rate 1.0 ml/min, gradient B20→60%, 20 min (data collection 30 min), column temperature 60°C, thermostat The temperature was 10° C., and the detector used two wavelengths of UV 225 nm (Ch1) and 280 nm (Ch2) for detection.

Samples a to f were reacted under the following conditions.
a: Trastuzumab;
b: Trastuzumab + 6 equivalents of peptide reagent;
c: reduction of trastuzumab with DTT;
d: reduction with D,L-dithiothreitol at buffer pH 8.0, antibody concentration 72 μM;
e: After 3 hours of reoxidation of d with dehydroascorbic acid;
f: 24 hours after reoxidation of d with dehydroascorbic acid.

It reacted in the order of b, d, and e or f, and after decomposing into light chain and heavy chain (d), it was reoxidized by dehydroascorbic acid to become e or f (Fig. 7). In addition, from the peak retention time, the light chain of trastuzumab and the light chain of the modified product appear at the same position, and the heavy chain retention time is shifted, indicating that the heavy chain is modified ( Figure 7).

(6-3) Linker Cleavage of Rituximab/Peptide Complex and Analysis by MALDI-TOFMS 400 μg of the rituximab/peptide complex synthesized in (5-3) of Example 5 was dissolved in 60 mM phosphate buffer (pH 8.0). , 72 μM, 10.0 μL of 20 mM D,L-dithiothreitol (60 equivalents to the rituximab-peptide complex) was added and stirred at room temperature for 16 hours. The solvent of the reaction solution was replaced with 9.57 mM PBS buffer (pH 7.0) using Amicon 10K to adjust the concentration to 18 μM, and then 6.4 μL of 10 mM dehydroascorbic acid (20 equivalents to the rituximab-peptide complex) was added. The mixture was stirred at room temperature for 3 hours to break the disulfide bond in the linker. When the mass was measured by MALDI-TOFMS, a peak was observed at m/z 144783 where two thiopropionyl groups were introduced into the product.

[Example 7: Fluorescent labeling of thiol-introduced trastuzumab and rituximab and calculation of fluorescent labeling rate]
(7-1) Fluorescent Labeling of Trastuzumab Thiol Transducer and Calculation of Fluorescent Labeling Rate The trastuzumab thiol transductant synthesized in (6-1) of Example 6 was dissolved in 20 mM PBS buffer to a concentration of 0.3 mM. After that, 20 μL of DyLight550 (0.94 mM, dissolved in DMF) manufactured by Thermo Fisher was added and stirred at room temperature for 12 hours. After the reaction, the fluorescent dye was removed with a Dy Removal Column. Absorbance at 280 nm and 557 nm was measured with a BECKMAN COULTER DU 800 SPECTROPHOTOMETER. The fluorescence labeling rate was calculated as 2 from the absorbance.

(7-2) Fluorescent labeling of rituximab thiol derivative and calculation of fluorescent labeling rate The rituximab thiol derivative synthesized in (6-3) of Example 6 was dissolved in 20 mM PBS buffer to a concentration of 0.3 mM. After that, 20 μL of DyLight550 (0.94 mM, dissolved in DMF) manufactured by Thermo Fisher was added and stirred at room temperature for 12 hours. After the reaction, the fluorescent dye was removed with a Dy Removal Column. BECKMAN
Absorbance at 280 nm and 557 nm was measured with a COULTER DU 800 SPECTROPHOTOMETER. The fluorescence labeling rate was calculated as 2 from the absorbance.

[Example 8: Peptide mapping of IgG1 Fc and thiol derivatives of trastuzumab]
(8-1) IgG1 Fc and trastuzumab, trypsin treatment of rituximab thiol introducer Dispense 40 μL of 100 mM ammonium bicarbonate buffer containing 8 M urea into a 1.5 mL low-adsorption microtest tube, sample solution 12 μL, 100 mM carbonate 2 μL of 2% ProteaseMAX (protease digestion accelerator ProteaseMAX ^™ Surfactant) dissolved in an ammonium hydrogen buffer solution was added, stirred and mixed. After that, 20 μL of 20 mM dithiothreitol aqueous solution was added and after heating at 65° C. for 15 minutes, 42 μL of 30 mM iodoacetic acid aqueous solution was added and reacted for 30 minutes at room temperature in the dark. After the reaction, 150 μL of 100 mM ammonium bicarbonate buffer was added and stirred, 1.4 μL of 2% ProteaseMAX dissolved in 100 mM ammonium bicarbonate buffer was added, and 100 μg/mL trypsin aqueous solution (Proteomics Grade, Code No. T6567- 5×20 μg (SIGMA)) was added to 10 μL and enzymatically digested at 37° C. for 18 hours. After digestion, 15 μL of 10% trifluoroacetic acid aqueous solution and 4.6 μL of 0.01% trifluoroacetic acid aqueous solution containing 2% acetonitrile were added and used for LC-MS/MS measurement.

(8-2) Glu-C treatment of IgG1 Fc and trastuzumab thiol-introducer Dispense 20 μL of 100 mM ammonium bicarbonate buffer containing 8 M urea into a 1.5 mL low-adsorption microtest tube, add 8 μL of the sample solution, and stir. , mixed. Then, 5 μL of 100 mM dithiothreitol aqueous solution was added, and after heating at 65° C. for 15 minutes, 6 μL of 200 mM iodoacetic acid aqueous solution was added, and the reaction was allowed to proceed for 30 minutes at room temperature in the dark. After the reaction, 2 μL of 100 mM dithiothreitol aqueous solution is added, and 3 μL of 100 mM ammonium hydrogen carbonate buffer is added and stirred, and 200 μg/mL Glu-C Protease aqueous solution (Pierce ^™ Glu-C Protease, MS Grade, product number :90054) was added and enzymatically digested at 37°C for 18 hours. After digestion, 4 μL of 10% trifluoroacetic acid aqueous solution was added and used for LC-MS/MS measurement.

(8-3) IgG1 Fc and LC-MS/MS measurement conditions for trastuzumab (analyzer)
Nano HPLC: EASY-nLC 1000 (Thermo Fisher Scientific)
Mass spectrometer: Tribrid mass spectrometer Orbitrap Fusion (Thermo Fisher Scientific)

(HPLC analysis conditions)
Trap column: Acclaim PepMap® 100, 75 μm×2 cm, nanoViper (Thermo Fisher Scientific)
Analysis column: ESI-column (NTCC-360/75-3-125, 75 μm×12.5 cm, 3 μm (Nikkyo Technos Co., Ltd.))
Mobile phase A: 0.1% formic acid aqueous solution Mobile phase B: 0.1% formic acid, acetonitrile solution Loading solution: 0.1% trifluoroacetic acid aqueous solution Flow rate: 300 nL/min
Sample injection volume: 2 μL (IgG1 Fc), 3 μL (trastuzumab)
Gradient conditions (B%): 2% (0.0-0.5 minutes), 2% → 45% (0.5-33.5 minutes), 45% → 75% (33.5-35.5 minutes ), 75% (35.5-45.0 min)

(Mass spectrometer analysis conditions)
Ionization method: ESI, Positive mode Scan type: Data Dependent Aquisition
Activation Type: Collision Induced Dissociation (CID)
Acquisition of data was carried out using attached software Xcalibur 3.0 (Thermo Fisher Scientific) and Thermo Orbitrap Fusion Tune Application 2.0 (Thermo Fisher Scientific).

(8-4) Analysis Conditions for Modified Sites of IgG1 Fc and Trastuzumab Modified site analysis for LC-MS/MS measurement results was performed using Proteome Discoverer version 1.4 (Thermo Fisher Scientific).

Analyzes on Proteome Discoverer used Sequest HT as the search engine with a precursor ion range of 350-5000 Da. As for the digestive enzyme, trypsin or Glu-C was set as the digestive enzyme according to the sample, and Maximum Missed Cleavage Sites was set to 0. Also, the mass tolerances of precursor and fragment ions were set to 10 ppm and 0.5 Da, respectively. Carboxymethyl (+58.005 Da) was set as Static Modification as modification of cysteine residue with iodoacetic acid. For Dynamic Modifications, oxidation of methionine (+15.995 Da) and modifications to lysine residues (thiol-introduced carboxymethylated with iodoacetic acid (+146.004 Da) were set.

In addition, (1) to (3) shown in FIG. 8 were used as the amino acid sequence data to be searched for the modification site.

(8-5) IgG1 Fc and trastuzumab modification site analysis results by LC-MS/MS As a result of analysis using LC/MS/MS, modification sites to lysine residues by trypsin digestion of trastuzumab (Carboxymethyl by iodoacetic acid MS spectrum of a peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPK (SEQ ID NO: 5) consisting of 26 amino acid residues containing a thiol-introduced thiol (+146.004 Da)) (actual value: m / z 998.14874, theoretical value: 998 .14859, trivalent) was observed (Fig. 9), and m / z 1375.59 (theoretical value : 1375.14) was confirmed (Fig. 10). In addition, a peptide consisting of 16 amino acid residues containing a modification site to a lysine residue by Glu-C Protease digestion of trastuzumab (thiol-introduced carboxymethylated with iodoacetic acid (+146.004 Da)), LLGGPSVFLFPPKPKD (SEQ ID NO: 6 ) of the peptide fragment (actual value: m / z 929.49573, theoretical value: 929.49527, bivalent) was observed (Fig. 11), and from the CID spectrum, the lysine residue at position 248 in EU numbering of the heavy chain A product ion with m/z 505.22 (theoretical value: 505.20) corresponding to divalent y3, indicating modification of the group, was confirmed (Fig. 12).

Furthermore, THTCPPCPAPEAEGAPSVFLFPPKPK (SEQ ID NO: 7), a peptide consisting of 26 amino acid residues containing a modification site to a lysine residue by tryptic digestion of IgG1 Fc (thiol-introduced carboxymethylated with iodoacetic acid (+146.004 Da)). The MS spectrum of the peptide fragment (actual value: m / z 994.12885, theoretical value: 994.12433, trivalent) was observed (Fig. 13), and from the CID spectrum, the lysine residue at position 246 in EU numbering of the heavy chain A product ion with m/z 1369.16 (theoretical value: 1369.10) corresponding to divalent b24, indicating the modification, was confirmed (Fig. 14). In addition, a peptide consisting of 23 amino acid residues containing a site for modification of lysine residues by Glu-C Protease digestion of IgG1 Fc (thiol-introduced carboxymethylated by iodoacetic acid (+146.004 Da)), GAPSVFLFPPKPKDTLMISRTPE (SEQ ID NO: The MS spectrum of the peptide fragment of 8) (actual value: m / z 892.12604, theoretical value: 892.12611, trivalent) was observed (Fig. 15), and from the CID spectrum, the lysine at position 248 in the EU numbering of the heavy chain A product ion with m/z 620.27 (theory: 619.85) corresponding to divalent b12 was identified, indicating a residue modification (Figure 16).
From the above, modification to peptide fragments containing lysine residues at positions 246 and 248 in EU numbering was confirmed (FIGS. 9 to 16).

Example 9 Synthesis of Regioselective Trastuzumab-DM1 Conjugates and Analysis of Average DAR
(9-1) Synthesis of regioselective T-DM1* (trastuzumab-DM1) conjugate 200 μL of 0.35 mg of the compound having two bioorthogonal functional groups synthesized in (6-1) of Example 6 100 mM PBS, 10 mM EDTA buffer to 1.75 mg/mL, then 10 μL of N,N'-dimethylformamide and DM-1 (manufactured by Abzena) were dissolved in N,N'-dimethylformamide to 5 mM. 4.8 μL of the above was added and stirred at 20° C. for 2 hours. After 2 hours, 1 μL of N-acetylcysteine dissolved in 100 mM PBS and 10 mM EDTA buffer to 50 mM was added, and the mixture was further stirred at 20° C. for 1 hour. After the reaction, extraction was performed using a NAP-5 column to remove DM-1.

(9-2) Measurement of Antibody Concentration after Reaction with SoloVPE Absorbance was measured at a wavelength of 280 nm, and the antibody concentration was calculated from the molar extinction coefficient to be 0.48 mg/mL. Since the solution volume was 0.7 mL, the yield can be calculated as 0.336 mg.

(9-3) Calculation of DAR by Q-TOFMS Q-TOFMS is Agilent 6550 coupled to
An Agilent 1290 UPLC with DAD detector, Autosampler cooling and Thermostatted column compartment was used. DAR was calculated using MassHunter software. As for the product, a peak was observed at 149945 where two DM-1s were introduced, and the average DAR was calculated to be 2 (Fig. 23).

Ｃｈｅｍｉｃａｌ Ｆｏｒｍｕｌａ： Ｃ４６Ｈ６０ＣｌＮ５Ｏ１３
Ｅｘａｃｔ Ｍａｓｓ： ９２５．３８７６ DM1* used in this example is as follows.

Chemical Formula: C46H60ClN5O13
Exact Mass: 925.3876

[Example 10: Binding evaluation by Biacore]
(10-1) Evaluation of binding property to antigen HER2 by ELISA The binding property of the regioselective T-DM1 (trastuzumab-DM1) conjugate synthesized in (9-1) of Example 9 to antigen HER2 was evaluated by ELISA. bottom. Kadcyla (T-DM1) was also evaluated as a control. As a result, the dissociation constant (KD value) of Kadcyla was 6.372±0.76 pM and that of the regioselective T-DM1 (trastuzumab-DM1) conjugate was 6.684±0.17 pM. Therefore, it is considered that the ADC synthesized by this method maintains antigen-binding ability.

(10-2) Binding evaluation to FcRn by Biacore The binding to the neonatal Fc receptor (FcRn) of the regioselective T-DM1 (trastuzumab-DM1) conjugate synthesized in (9-1) of Example 9 was evaluated. Evaluated by ELISA. It is known that antibodies have a longer blood half-life when they have higher binding to FcRN. Kadcyla (T-DM1) was also evaluated as a control. As a result, the dissociation constant (KD value) of Kadcyla was 1.65±0.05 μM and that of the regioselective T-DM1 (trastuzumab-DM1) conjugate was 0.258±0.001 μM. Therefore, it is considered that the ADC synthesized by this method maintains FcRn-binding ability, and it was found that the affinity to FcRn is about 6 times higher than that of the existing ADC Kadcyla.

[Example 11: (1) Synthesis of various cleavable linkers and (2) Linking of synthesized linkers to IgG1 Fc-binding peptides with affinity substances, cleavable moieties and reactive groups for soluble proteins Compounds (peptide-azido modified thioester linker conjugate-NHS activated, peptide-maleimide modified thioester linker conjugate-NHS activated, peptide-azido modified ester linker conjugate-NHS activated, and peptide acetal linker conjugate -NHS activated form) synthesis]
(11-1) Synthesis of thioester linker and linking with peptide (11-1-1) Synthesis of thioester linker

6-Azidohexanoic acid (300 mg, 1.91 mmol) was dissolved in THF solvent (20 mL), and isobutyl chlorocarbonate (284 μL, 2.10 mmol) and N-methylmorpholine (273 μL, 2.48 mmol) were added at 0°C. After stirring for 30 minutes, a mixed anhydride of 6-azidohexanoic acid was prepared. After dissolving 4-(tert-butoxy-oxo)-2-amino-butanoic acid (362 mg, 2.10 mmol) in 1 M aqueous sodium hydroxide solution (2.10 mL) at room temperature, the above 6-azidohexanoic acid of the mixed acid anhydride in THF was added dropwise at room temperature. After stirring at room temperature for 16 hours, a 6M hydrochloric acid aqueous solution was added to adjust the pH of the system to 3.0. The reaction mixture was diluted with ethyl acetate, washed with water and brine, added with anhydrous magnesium sulfate, and allowed to stand for 5 minutes. After removing magnesium sulfate by filtration and concentrating under reduced pressure to obtain a crude product, it was purified by column chromatography (dichloromethane:methanol=10:1). Fractions containing the product were collected and concentrated under reduced pressure to give 4-(tert-butoxy-oxo)-2-(6-azidohexy-1-oxo)amino-butanoic acid (352 mg, 1.07 mmol). Obtained.

¹ H NMR (400 MHz, Chloroform-d) δ 6.56 (d, J=7
. 6Hz, 1H), 4.76 (ddd, J = 7.6, 5.2, 4.8Hz, 1H), 3.22 (t, 3.6Hz, 2H), 2.90 (dd, J = 17 .2, 4.8 Hz, 1 H), 2.70 (dd, J=17.2, 5.2 Hz, 1 H), 2.22 (t, J=7.2 Hz, 2 H), 1.53-1. 65 (m, 4H), 1.37 (s, 9H), 1.36-1.31 (m, 2H).
MS (ESI) m/z: 351 [M+Na] ⁺

4-(tert-butoxy-oxo)-2-(6-azidohexy-1-oxo)amino-butanoic acid (50.0 mg, 0.152 mmol) was dissolved in THF (1.5 mL) and chloroformed at 0°C. Isobutyl carbonate (30.9 μL, 0.228 mmol) and N-methylmorpholine (28.5 μL, 0.258 mmol) were added and stirred for 30 minutes to prepare the corresponding mixed acid anhydride. After dissolving mercaptopropionic acid (32.3 mg, 0.456 mmol) in THF (ml) at room temperature, the above THF solution of mixed acid anhydride was added dropwise at room temperature. After stirring at room temperature for 16 hours, 1M sodium hydroxide aqueous solution was added to adjust the pH of the system to 9.0. The reaction solution was washed with water and ethyl acetate, and the aqueous phase was recovered. A 6M hydrochloric acid aqueous solution was added to the aqueous phase to adjust the pH of the system to 3.0, followed by liquid separation and extraction with ethyl acetate. placed. 3-[4-(tert-butoxy-oxo)-2-(6-azidohexy-1-oxo)amino-1-oxo]sulfanyl-propanoic acid was obtained by removing magnesium sulfate by filtration and concentrating under reduced pressure. (60.1 mg, 0.144 mmol) was obtained.

¹ H NMR (400 MHz, Chloroform-d) δ 6.69 (d, J = 9.6 Hz, 1 H), 4.89 (dt, J = 9.6, 4.4 Hz, 1 H), 3.22 (t , 8.5Hz, 2H), 3.07 (t, 7.2Hz, 2H), 2.82-2.60 (m, 3H), 2.29-2.22 (m, 3H), 1.70 −1.52 (m, 4H), 1.42-1.39 (m, 2H), 1.40 (s, 9H).
MS (ESI) m/z: 439 [M+H] ⁺

上記アミノ酸配列は、配列番号３９である。 (11-1-2) Linking of thioester linker and peptide

The amino acid sequence is SEQ ID NO:39.

The peptide (20.0 mg, 9.64 μmol) of Ac-RGNCAYHKGQLVWCTYH-NH ₂ (SEQ ID NO: 39) synthesized in Example 1-2, provided that two cysteines at positions 4 and 14 each have intramolecular disulfide bonds. ) was dissolved in 1 mL of N,N′-dimethylformamide and 3-[4-(tert-butoxy-oxo)-2-(6-azidohexy-1-oxo)amino-1-oxo]- Sulfanyl-propanoic acid (60.1 mg, 0.144 mmol), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (25.9 mg, 0.135 mmol), 1-hydroxybenzotriazole (18.2 mg, 0.135 mmol) was dissolved in 1 mL of DMF and added into the system. After stirring at room temperature for 12 hours, 0.1% trifluoroacetic acid aqueous solution was added and elution was performed by reverse phase preparative chromatography. Fractions containing the product were collected and concentrated under reduced pressure to remove only acetonitrile, followed by freeze-drying to obtain the peptide linker-linked-tBu form (15 mg, 7.23 μmol).

MS (ESI) m/z: z=2 1237 [M+2H] ^<2+> , z=3 824 [M+3H] ^<3+>

上記アミノ酸配列は、配列番号３９である。

The amino acid sequence is SEQ ID NO:39.

The above peptide linker conjugate (10.0 mg, 4.82 μmol) was dissolved in 0.250 mL of dichloromethane, 0.250 mL of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 1 hour. After concentrating under reduced pressure, 0.1% trifluoroacetic acid aqueous solution was added and elution was performed by reverse phase preparative chromatography. Fractions containing the product were collected and concentrated under reduced pressure to remove only acetonitrile, followed by freeze-drying to obtain the peptide linker conjugate-carboxylic acid form (6.2 mg, 2.57 μmol).

MS (ESI) m/z: z=2 1208 [M+2H] ^<2+> , z=3 806 [M+3H] ^<3+>

上記アミノ酸配列は、配列番号３９である。

The amino acid sequence is SEQ ID NO:39.

The peptide linker conjugate-carboxylic acid form (5.7 mg, 2.36 μmol) was
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (9.1 mg, 47.2 μmol), N-hydroxysuccinimide (8.1 mg, 70.8 μmol) was dissolved in 0.5 mL of N′-dimethylformamide. ) was added and stirred for 16 hours. A 0.1% trifluoroacetic acid aqueous solution was added to the reaction mixture, and the mixture was eluted by reverse phase preparative chromatography. Fractions containing the product were collected and concentrated under reduced pressure to remove only acetonitrile, followed by freeze-drying to obtain the peptide-azido-modified thioester linker conjugate-NHS activated form (3.0 mg, 1.19 μmol). got

MS (ESI) m/z: z=2 1257 [M+2H] ^<2+> , z=3 838 [M+3H] ^<3+>

上記アミノ酸配列は、配列番号３９である。

The amino acid sequence is SEQ ID NO:39.

The peptide-azido-modified thioester linker conjugate-NHS activator (3.0 mg, 1.20 μmol) was dissolved in 0.5 mL of N,N′-dimethylformamide, and the dibenzocyclooctyne-maleimide (0.6 mg, 1 .2 μmol) was added and stirred for 16 hours. A 0.1% trifluoroacetic acid aqueous solution was added to the reaction mixture, and the mixture was eluted by reverse phase preparative chromatography. Fractions containing the product were collected and concentrated under reduced pressure to remove only acetonitrile, followed by freeze-drying to give the above peptide-maleimide-modified thioester linker conjugate-NHS activated form (2.8 mg, 0.95 μmol). got

MS (ESI) m/z: z=2 1471 [M+2H] ^<2+> , z=3 981 [M+3H] ^<3+>

(11-2) Synthesis of ester linker and linking with peptide (11-2-1) Synthesis of ester linker

4-(tert-butoxy-oxo)-2-[(6-azidohexy-1-oxo)amino]-butanoic acid (50 mg, 0.152 mmol) was dissolved in THF (1.5 mL) and chloroformed at 0°C. Isobutyl carbonate (22.7 μL, 0.167 mmol) and N-methylmorpholine (21.7 μL, 0.198 mmol) were added and stirred for 30 minutes to prepare the corresponding mixed acid anhydride. After dissolving sodium borohydride (28.7 mg, 0.76 mmol) in 1M sodium hydroxide aqueous solution (1.0 ml) at room temperature, the THF solution of the above mixed acid anhydride was added dropwise at room temperature. After stirring at room temperature for 16 hours, ethyl acetate was added to the system, and after washing with water and brine, the organic phase was recovered, anhydrous magnesium sulfate was added, and the mixture was allowed to stand for 5 minutes. After removing magnesium sulfate by filtration and concentrating under reduced pressure to obtain a crude product, it was purified by column chromatography (dichloromethane:methanol=10:1). Fractions containing the product were collected and concentrated under reduced pressure to give tert-butyl 4-hydroxy-3-[(6-azidohexy-1-oxo)amino]-butanoate (38.1 mg, 0.121 mmol). Obtained.

¹ H NMR (400 MHz, Chloroform-d) δ 6.36 (d, J=7.2 Hz, 1 H), 4.20 (m, 1 H), 3.69 (dt, 12.4, 4.4 Hz, 2 H ), 3.27 (t, J = 6.8 Hz, 2H), 2.50 (d, J = 6.0 Hz, 2H), 2.22 (t, J = 7.2 Hz, 2H), 1.53 −1.72 (m, 4H), 1.41 (s, 9H), 1.40-1.38 (m, 2H).
MS (ESI) m/z: 337 [M+Na] ⁺

tert-Butyl-4-hydroxy-3-[(6-azidohexy-1-oxo)amino]-butanoate (70.4 mg, 0.224 mmol) was dissolved in THF (2.2 mL) and succinic anhydride was added at room temperature. (67.3 mg, 0.673 mmol) and N,N-dimethylaminopyridine (82.2 mg, 0.673 mmol) were added and stirred at room temperature for 16 hours. The pH was adjusted to 9.0. After water and ethyl acetate were added to the reaction liquid and liquid separation was performed, the aqueous phase was recovered. A 6M hydrochloric acid aqueous solution was added to the aqueous phase to adjust the pH of the system to 3.0, followed by liquid separation and extraction with ethyl acetate. placed. 4-[4-(tert-butoxy-oxo)-2-(6-azidohexy-1-oxo)amino]oxy-4-oxo-propanoic acid was obtained by removing magnesium sulfate by filtration and concentrating under reduced pressure. (78.9 mg, 0.190 mmol) was obtained.

¹ H NMR (400 MHz, Chloroform-d) δ 6.28 (d, J = 8.0 Hz, 2H), 4.52 (m, 1H), 4.29 (dd, J = 11.2, 4.4 , 1H), 4.15 (dd, J = 11.2, 5.6), 3.28 (m, 2H), 2.66 (m, 4H), 2.49 (d, J = 6.4Hz , 2H), 2.18 (t, J = 7.2, 2H), 1.72-1.50 (m, 4H), 1.42 (s, 9H), 1.41-1.38 (m , 2H).
MS (ESI) m/z: 415 [M+H] ⁺

上記アミノ酸配列は、配列番号３９である。 (11-2-2) Linking of ester linker and peptide

The amino acid sequence is SEQ ID NO:39.

The peptide (20 mg, 9.63 μmol) of Ac-RGNCAYHKGQLVWCTYH-NH ₂ (SEQ ID NO: 39) synthesized in Example 1-2, provided that the two cysteines at the 4th and 14th positions each form an intramolecular disulfide bond. ) was dissolved in 1 mL of N,N′-dimethylformamide, and (4-[4-(tert-butoxy-oxo)-2-(6-azidohexy-1-oxo)amino]oxy-4-oxo- Propanoic acid (39.9 mg, 96.3 μmol), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (25.8 mg, 0.135 mmol), 1-hydroxybenzotriazole (18.2 mg, 0.135 mmol). 135 mmol) was dissolved in 1 mL of DMF and added into the system.After stirring at room temperature for 12 hours, 0.1% trifluoroacetic acid aqueous solution was added and eluted by reverse phase preparative chromatography.The fraction containing the product was collected. After removing only acetonitrile by concentrating under reduced pressure, lyophilization was performed to obtain the peptide linker-linked-tBu derivative (18.2 mg, 7.36 μmol).

MS (ESI) m/z: z=2 1236 [M+2H] ^<2+> , z=3 824 [M+3H] ^<3+>

上記アミノ酸配列は、配列番号３９である。

The amino acid sequence is SEQ ID NO:39.

The above peptide linker conjugate (16.5 mg, 6.67 μmol) was dissolved in 1.0 mL of dichloromethane, 1.0 mL of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 1 hour. After concentrating under reduced pressure, 0.1% trifluoroacetic acid aqueous solution was added and elution was performed by reverse phase preparative chromatography. Fractions containing the product were collected and concentrated under reduced pressure to remove only acetonitrile, followed by freeze-drying to obtain the peptide linker conjugate-carboxylic acid form (15.2 mg, 6.29 μmol).

MS (ESI) m/z: z=2 1208 [M+2H] ^<2+> , z=3 805 [M+3H] ^<3+>

上記アミノ酸配列は、配列番号３９である。

The amino acid sequence is SEQ ID NO:39.

The peptide linker conjugate-carboxylic acid form (3.0 mg, 1.21 μmol) was dissolved in 1 mL of N,N'-dimethylformamide, and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (4. 7 mg, 24.3 μmol) and N-hydroxysuccinimide (4.2 mg, 36.4 μmol) were added and stirred for 16 hours. A 0.1% trifluoroacetic acid aqueous solution was added to the reaction mixture, and the mixture was eluted by reverse phase preparative chromatography. Fractions containing the product are collected and concentrated under reduced pressure to remove only acetonitrile, followed by freeze-drying to obtain the peptide-azido modified ester linker conjugate-NHS activated product (3.0 mg, 1.19 μmol). got

MS (ESI) m/z: z=2 1257 [M+2H] ^<2+> , z=3 837 [M+3H] ^<3+>

(11-3) Synthesis of acetal linker and ligation of peptide (11-3-1) Synthesis of acetal linker

3,9-bis(2-cyanoethyl)-2,4,8,10-[5.5]tetraoxaspiroundecane (200 mg, 0.752 mmol) was dissolved in 20 mL of 8 M aqueous sodium hydroxide solution and stirred at 100° C. for 16 Stirred for hours. After 16 hours, the temperature was returned to room temperature, and a 6M hydrochloric acid aqueous solution was added to the aqueous phase to adjust the pH of the system to 7.0. After recovering the acetonitrile phase, anhydrous sodium sulfate was added and the mixture was allowed to stand for 5 minutes. Sodium sulfate was removed by filtration and concentration under reduced pressure gave 2,4,8,10-[5.5]tetraoxaspironedecane-3,9-dipropanoic acid (151 mg, 0.497 mmol). .

MS (ESI) m/z: 327 [M+Na] ⁺

2-[9-(2-Methoxy-2-oxo-ethyl)-2,4,8,10-tetraoxospiro[5.5]undecane-3-yl]acetic acid (30.0 mg, 0.099 mmol) It was dissolved in 0.5 mL of ethyl acetate, N,N'-dicyclohexylcarbodiimide (22.4 mg, 0.109 mmol) and N-hydroxysuccinimide (31.2 mg, 0.271 mmol) were added, and the mixture was stirred for 2 hours. The resulting white crystals were filtered, and the mother liquor was concentrated under reduced pressure to give bis(2,5-dioxopyrrolidin-1-yl)-2,4,8,10-[5.5]tetraoxaspironedecane-3,9- Dipropanoate (15 mg, 31.8 μmol) was obtained.

MS (ESI) m/z: 521 [M+Na] ⁺

上記アミノ酸配列は、配列番号３９である。 (11-3-2) Linking of acetal linker and peptide

The amino acid sequence is SEQ ID NO:39.

The peptide (3.0 mg, 1.45 μmol) of Ac-RGNCAYHKGQLVWCTYH-NH ₂ (SEQ ID NO: 39) synthesized in Example 1-2, provided that two cysteines at positions 4 and 14 each have intramolecular disulfide bonds. ) was dissolved in 0.3 mL of N,N′-dimethylformamide, and (bis(2,5-dioxopyrrolidin-1-yl)2,4,8,10-[5.5]tetra Oxaspironedecane-3,9-dipropanoate (14.4 mg, 28.9 μmol) was added and the mixture was stirred at room temperature for 12 hours, then water was added and eluted by reverse phase preparative chromatography using neutral acetonitrile and water as solvents. The fraction containing the product was collected and concentrated under reduced pressure to remove only acetonitrile, followed by freeze-drying to obtain the peptide acetal linker conjugate-NHS activated product (2.8 mg, 1.14 μmol).

MS (ESI) m/z: z=2 1729 [M+2H] ^<2+> , z=3 819 [M+3H] ^<3+>

[Example 12: Modification of anti-HER2 IgG antibody trastuzumab with various compounds and analysis by ESI-TOFMS]
(12-1) Specific modification of IgG antibody trastuzumab with azide-modified thioester linker-binding peptide reagent and analysis by ESI-TOFMS Peptide synthesized in (11-1-2) of Example 11-azido-modified thioester linker conjugate- The activated NHS was dissolved in N,N'-dimethylformamide to 4 mM. Dissolve 120 μg of anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical Co., Ltd.) in 39.8 μL of 50 mM sodium acetate buffer (pH 5.5), add 2.4 μL of 4 mM peptide reagent (12 equivalents to the antibody), and incubate at room temperature for 1 hour. Stirred for hours. The solvent of the reaction solution was replaced with 9.57 mM PBS buffer (pH 7.4) using Amicon 10K to terminate the reaction. When the mass was measured by ESI-TOFMS, a peak was observed at 148225 for the raw material trastuzumab, and a peak was observed at 153018 for the product into which two binding peptides were introduced.

(12-2) Confirmation of heavy chain selectivity by ESI-TOFMS analysis under reducing conditions of a specific modification of the anti-HER2 IgG antibody trastuzumab 7 mM Tris ( 2.0 μL of 2-carboxyethyl)phosphine hydrochloride solution (100 equivalents to the antibody) was added and stirred at room temperature for 20 minutes. When the mass was measured by ESI-TOFMS, the raw material trastuzumab had heavy chain peaks at 50595 and 50757 and a light chain peak at 23440. A light chain peak was observed at 23440, the same as the starting material.

(12-3) Specific modification of IgG antibody trastuzumab with maleimide-modified thioester linker-binding peptide reagent and analysis by ESI-TOFMS Peptide synthesized in (11-1-2) of Example 11-maleimide-modified thioester linker conjugate- The activated NHS was dissolved in N,N'-dimethylformamide to 4 mM. Dissolve 120 μg of anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical Co., Ltd.) in 39.8 μL of 50 mM sodium acetate buffer (pH 5.5), add 2.4 μL of 4 mM peptide reagent (12 equivalents to the antibody), and incubate at room temperature for 1 hour. Stirred for hours. The solvent of the reaction solution was replaced with 9.57 mM PBS buffer (pH 7.4) using Amicon 10K to terminate the reaction. When the mass was measured by ESI-TOFMS, a peak was observed at 148225 for the raw material trastuzumab, and a peak was observed at 153901 for the product into which two binding peptides were introduced.

(12-4) Confirmation of heavy chain selectivity by ESI-TOFMS analysis under reducing conditions of a specific modification of the anti-HER2 IgG antibody trastuzumab 7 mM Tris ( 2.0 μL of 2-carboxyethyl)phosphine hydrochloride solution (100 equivalents to the antibody) was added and stirred at room temperature for 20 minutes. When the mass was measured by ESI-TOFMS, the raw material trastuzumab had heavy chain peaks at 50594 and 50757, and a light chain peak at 23440. A light chain peak was observed at 23440, the same as the starting material.

(12-5) Specific modification of IgG antibody trastuzumab with azide-modified ester linker-binding peptide reagent and analysis by ESI-TOFMS Peptide synthesized in (11-2-2) of Example 11-azido-modified ester linker conjugate- The activated NHS was dissolved in N,N'-dimethylformamide to 4 mM. Dissolve 120 μg of anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical Co., Ltd.) in 39.8 μL of 50 mM sodium acetate buffer (pH 5.5), add 2.4 μL of 4 mM peptide reagent (12 equivalents to the antibody), and incubate at room temperature for 1 hour. Stirred for hours. The solvent of the reaction solution was replaced with 9.57 mM PBS buffer (pH 7.4) using Amicon 10K to terminate the reaction. When the mass was measured by ESI-TOFMS, a peak was observed at 148225 for the raw material trastuzumab, and a peak was observed at 153028 for the product into which two binding peptides were introduced.

(12-6) Confirmation of heavy chain selectivity by ESI-TOFMS analysis under reducing conditions of a specific modification of the anti-HER2 IgG antibody trastuzumab 7 mM Tris ( 2.0 μL of 2-carboxyethyl)phosphine hydrochloride solution (100 equivalents to the antibody) was added and stirred at room temperature for 20 minutes. When the mass was measured by ESI-TOFMS, the raw material trastuzumab had heavy chain peaks at 50594 and 50757 and a light chain peak at 23440, and the product had peaks at 52994 and 53154 where a binding peptide was introduced into the heavy chain. Confirmed, a light chain peak was observed at 23440, the same as the starting material.

(12-7) Specific modification of IgG antibody trastuzumab with acetal linker-binding peptide reagent and analysis by ESI-TOFMS Peptide synthesized in (11-3-2) of Example 11-acetal linker conjugate-NHS activated product was dissolved in N,N'-dimethylformamide to 4 mM. 120 μg of anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical Co., Ltd.) in 50 mM sodium acetate buffer (pH
5.5) Dissolved in 39.8 μL, added 2.4 μL of 4 mM peptide reagent (12 equivalents to the antibody), and stirred at room temperature for 1 hour. The solvent of the reaction solution was replaced with 9.57 mM PBS buffer (pH 7.4) using Amicon 10K to terminate the reaction. When the mass was measured by ESI-TOFMS, a peak was observed at 148238 for the raw material trastuzumab, and a peak was observed at 153522 for the product into which two binding peptides were introduced.

(12-8) Confirmation of heavy chain selectivity by ESI-TOFMS analysis under reducing conditions of a specific modification of anti-HER2 IgG antibody trastuzumab 7 mM Tris ( 2.0 μL of 2-carboxyethyl)phosphine hydrochloride solution (100 equivalents to the antibody) was added and stirred at room temperature for 20 minutes. When the mass was measured by ESI-TOFMS, the raw material trastuzumab had heavy chain peaks at 50595 and 50757 and a light chain peak at 23440. A light chain peak was observed at 23440, the same as the starting material.

Example 13 Cleavage of Trastuzumab Peptide Complexes and Analysis of Their Products by ESI-TOFMS
(13-1) Linker Cleavage of Trastuzumab-Peptide Complex and ESI-TOFMS Analysis of Product pH 8.5) to 2.0 μM, and stirred at room temperature for 48 hours to cleave the thioester bond in the linker. ESI-TOF
When the mass was measured by MS, a peak was observed at 153018 for the trastuzumab-peptide complex, and a peak was observed at 148729 for the product into which two azides were introduced.

(13-2) Confirmation of heavy chain selectivity by ESI-TOFMS analysis under reducing conditions of a specific modification of trastuzumab 7 mM tris(2-carboxyethyl)phosphine on the antibody/peptide complex produced in (13-1) 2.0 μL of hydrochloride solution (100 equivalents to antibody) was added and stirred at room temperature for 20 minutes. When the mass was measured by ESI-TOFMS, the raw material trastuzumab peptide complex had heavy chain peaks at 52995 and 53155 and a light chain peak at 23440. A peak was observed and a light chain peak was observed at 23440, the same as the starting material.

(13-3) Linker Cleavage of Trastuzumab-Peptide Complex and ESI-TOFMS Analysis of Product pH 8.5) to 2.0 μM, and stirred at room temperature for 48 hours to cleave the thioester bond in the linker. When the mass was measured by ESI-TOFMS, a peak was observed at 153901 for the trastuzumab-peptide complex, and a peak was observed at 149476 for the product into which two maleimides were introduced.

(13-4) Confirmation of heavy chain selectivity by ESI-TOFMS analysis under reducing conditions of a specific modification of trastuzumab 7 mM tris(2-carboxyethyl)phosphine on the antibody/peptide complex produced in (13-3) 2.0 μL of hydrochloride solution (equal volume to antibody) was added and stirred at room temperature for 20 minutes. When the mass was measured by ESI-TOFMS, the trastuzumab peptide complex observed heavy chain peaks at 53421 and 53600, light chain peaks at 23440, and the product peaks at 51278 and 51439 where maleimide was introduced into the heavy chain. was observed, and a light chain peak was observed at 23440, the same as the starting material.

(13-5) Cleavage of ester linker and analysis by ESI-TOFMS Dissolve the antibody/peptide ester linker conjugate synthesized in (12-5) of Example 12 in any buffer (pH 4.5 to 9.0). Then, by adding an esterase enzyme and stirring at room temperature, the ester structure is hydrolyzed and the azide group remains on the antibody. The mass of this can be measured by ESI-TOFMS.

(13-6) Linker Cleavage of Trastuzumab-Peptide Complex and ESI-TOFMS Analysis of Product pH 2.0) to 2.0 μM, and then stirred at room temperature to cleave the acetal bond in the linker. When the mass was measured by ESI-TOFMS, a peak was observed at 1535223 for the trastuzumab-peptide complex, and a peak was observed at 148452 for the product into which two aldehydes were introduced.

(13-7) Confirmation of heavy chain selectivity by ESI-TOFMS analysis under reducing conditions of a specific modification of trastuzumab 7 mM tris(2-carboxyethyl)phosphine on the antibody/peptide complex produced in (13-1) 2.0 μL of hydrochloride solution (100 equivalents to antibody) was added and stirred at room temperature for 20 minutes. When the mass was measured by ESI-TOFMS, the starting antibody/peptide complex was observed to have heavy chain peaks at 52940 and 53103, and a light chain peak at 23440.
As for the product, peaks were observed at 50683 and 50845 where aldehyde was introduced into the heavy chain, and a light chain peak was observed at 23440 which was the same as the raw material.

[Example 14: Conjugation of trastuzumab-azide derivative and low molecular compound] (14-1) Conjugation of trastuzumab azide derivative and low molecular compound Synthesized in (13-1) of Example 13, After dissolving the azide derivative of trastuzumab in 20 mM PBS buffer to 0.3 mM, 20 μL of Sigma Aldrich's Dibenzocycloctyne-Cy3 (0.94 mM, dissolved in DMF) was added and stirred at room temperature for 12 hours. The solvent of the reaction solution was replaced with 9.57 mM PBS buffer (pH 7.4) using Amicon 10K to terminate the reaction. When the mass was measured by ESI-TOFMS, a peak was observed at 148706 for the azide-introduced trastuzumab starting material, and a peak was observed at 150688 for the product into which two low molecules were introduced.

(14-2) Confirmation of heavy chain selectivity by ESI-TOFMS analysis of trastuzumab-low molecular compound complex under reducing conditions 7 mM Tris (2 -Carboxyethyl)phosphine hydrochloride solution 2 μL (100 equivalents to the antibody) was added and stirred at room temperature for 20 minutes. When the mass was measured by ESI-TOFMS, the starting trastuzumab azide derivative had heavy chain peaks at 50,850 and 51,010 and a light chain peak at 23,440. A peak was observed at 23440, the same as the starting material, and a light chain peak was observed at 23440.

[Example 15: Peptide mapping of azide derivative of trastuzumab]
(15-1) Trypsin treatment of trastuzumab azide derivative (Example 14) 10 μL of sample solution in 1.5 mL low-adsorption microtest tube, 50 mM ammonium bicarbonate buffer, 20 mM dithio dissolved in 20% trifluoroethanol After adding 10 μL of threitol aqueous solution and heating at 65° C. for 1 hour, 10 μL of 50 mM iodoacetamide aqueous solution was added and reacted by shaking at 300 rpm for 30 minutes at room temperature in the dark. After the reaction, 40 μL of 50 mM ammonium bicarbonate buffer was added and stirred, and 10 μL of 20 ng/μL trypsin aqueous solution (Proteomics Grade, Code No. T6567-5×20 μg (SIGMA)) was added for enzymatic digestion at 37° C. for 18 hours. After digestion, 2 μL of 20% trifluoroacetic acid aqueous solution was added to stop the reaction. After that, it was diluted two-fold to an aqueous solution of trifluoroacetic acid with a final concentration of 0.25%, and used for LC-MS/MS measurement.

(15-2) Trastuzumab LC-MS/MS measurement conditions (analytical device)
Nano HPLC: EASY-nLC 1000 (Thermo Fisher Scientific)
Mass spectrometer: Tribrid mass spectrometer Orbitrap Fusion (Thermo Fisher Scientific)

(HPLC analysis conditions)
Trap column: Acclaim PepMap® 100, 75 μm x 2 cm (Thermo Fisher Scientific)
Analysis column: ESI-column (NTCC-360/75-3-125, 75 μm×12.5 cm, 3 μm (Nikkyo Technos Co., Ltd.))
Mobile phase A: 0.1% formic acid aqueous solution Mobile phase B: 0.1% formic acid, acetonitrile solution Loading solution: 0.1% trifluoroacetic acid aqueous solution Flow rate: 300 nL/min
Sample injection volume: 1 μL
Gradient conditions (B%): 2% (0.0-0.5 minutes), 2% → 30% (0.5-23.5 minutes), 30% → 75% (23.5-25.5 minutes ), 75% (25.5-35.0 min)

(Mass spectrometer analysis conditions)
Ionization method: ESI, Positive mode Scan type: Data Dependent Aquisition
Activation Type: Collision Induced Dissociation (CID)
Acquisition of data was carried out using attached software Xcalibur 3.0 (Thermo Fisher Scientific) and Thermo Orbitrap Fusion Tune Application 2.0 (Thermo Fisher Scientific).

(15-3) Analysis Conditions for Modified Site of Trastuzumab Modified site analysis for LC-MS/MS measurement results was performed using Proteome Discoverer version 1.4 (Thermo Fisher Scientific).

The analysis with Proteome Discoverer used Sequest HT as a search engine, with a precursor ion range of 350-5000 Da and a Total Intensity Threshold of 50000. Also, trypsin was set as a digestive enzyme, and Maximum Missed Cleavage Sites was set to 3. Also, the mass tolerances of precursor and fragment ions were set to 5 ppm and 0.5 Da, respectively. Carbamidomethyl (+57.021 Da) was set as Static Modification as modification of cysteine residue with iodoacetamide. For Dynamic Modifications, methionine oxidation (+15.995 Da) and modifications to lysine residues (azide introduction (+254.102 Da), amine introduction (+228.111 Da)) were set. Furthermore, a filter was applied so that Peptide Confidence was High only.

In addition, (1) to (3) shown in FIG. 24 were used as the amino acid sequence data to be searched for the modification site.

(15-4) Analysis results of modification site by LC-MS/MS of trastuzumab As a result of analysis using LC-MS/MS, modification site to lysine residue by trypsin digestion of trastuzumab (azide introduction (+254.102 Da )) containing 33 amino acid residues, a peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 40) MS spectrum (actual value: m / z 979.49982, theoretical value: 979.49975, tetravalent) was observed (Fig. 25), the product ion of m / z 1192.87 (theoretical value: 1192.64) corresponding to divalent y19, which indicates the modification of the lysine residue at position 246 in the EU numbering of the heavy chain from the CID spectrum, was confirmed. (Fig. 26).

[Example 16: Synthesis of a compound (peptide disulfide linker conjugate-NHS activator) having an affinity substance for soluble proteins, a cleavable moiety and a reactive group, and modification of the anti-HER2 IgG antibody trastuzumab using the compound and its analysis]
(16-1) Synthesis of IgG1 Fc-binding peptide The sequence of Ac-RGNCKYHRGQLVWCTYH-NH ₂ (SEQ ID NO: 42) was
c Synthesized by the solid-phase synthesis method. Liberty Blue manufactured by CEM was used as a peptide synthesizer. All reagents used are manufactured by Watanabe Chemical Industry. Resin was Fmoc-NH-SAL-PEG Resin HL, arginine (R), cysteine (C) and histidine (H) were double-coupled. Cutting from Resin was performed in a solution of trifluoroacetic acid:water:triisopropylsilane:ethanedithiol=94:2.5:1.0:2.5 under stirring conditions for 3 hours. After cutting out, the resin was removed by filtration, and the trifluoroacetic acid was removed. Diethyl ether was added to the resulting crystals for ether precipitation, and the resulting white crystals were collected by filtration. This was dissolved in an aqueous 0.05% trifluoroacetic acid solution and subjected to reversed-phase high performance liquid chromatography using octadodecyl chemically bonded silica gel as a packing material. Elution was performed with the mixed solution, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain the desired product (36.4 mg, 16.8 μmol).

MS (ESI) m/z: z=3 721.80 [M+3H] ^<3+> , z=4 541.60 [M+4H] ^<4+>

上記アミノ酸配列は、配列番号４２である。 (16-2) Formation of intramolecular disulfide bond at Cys at positions 4 and 14 of Ac-RGNCKYHRGQLVWCTYH-NH ₂ (SEQ ID NO: 42)

The amino acid sequence is SEQ ID NO:42.

The peptide (36.4 mg, 16.8 µmol) synthesized in (16-1) was dissolved in DMSO (5.00 mL), and 2M NH ₃ -MeOH (17.0 µL, 33.6 µmol), hydrogen peroxide solution (34. 0 μL, 0.336 mmol) was added and stirred at room temperature for 20 hours. A 2M trifluoroacetic acid aqueous solution was added to the reaction solution to terminate the reaction, dissolved in a 0.05% trifluoroacetic acid aqueous solution, and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. Elution was performed with a mixed solution of water and acetonitrile containing 0.05% fluoroacetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain the above peptide (20.3 mg, 9.40 μmol).

MS (ESI) m/z: z=3 721.25 [M+3H] ^<3+> , z=4 541.20 [M+4H] ^<4+>

上記アミノ酸配列は、配列番号４２である。 (16-3) Linking disulfide linker and peptide

The amino acid sequence is SEQ ID NO:42.

Ac-RGNCKYHRGQLVWCTYH-NH ₂ (SEQ ID NO: 42) (20.3 mg, 9.40 μmol) synthesized in (16-2), provided that two cysteines at positions 4 and 14 each form an intramolecular disulfide bond ) was dissolved in N,N-dimethylformamide (1.00 mL), and di(N-succinimidyl) 3,3′-dithiodipropionate (228 mg, 0.563 mmol) was dissolved in acetonitrile (2.00 mL). was added and stirred at room temperature for 24 hours. After removing acetonitrile by concentrating under reduced pressure, this was dissolved in 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. Elution was performed with a mixed solution of water and acetonitrile containing 0.05% acetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain the peptide disulfide linker conjugate-NHS activated product (6.80 mg, 2.78 μmol). .

MS (ESI) m/z: z=3 817.60 [M+3H] ^<3+> , z=4 613.45 [M+4H] ^<4+>
HPLC Purity: 100%

(16-4) Specific modification of anti-HER2 IgG antibody trastuzumab and analysis by MALDI-TOFMS The peptide disulfide linker conjugate-NHS-activated product synthesized in (16-3) was dissolved in N,N'-dimethylformamide. 6 mM. Dissolve 500 μg of anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical Co., Ltd.) in 46.9 μL of 60 mM sodium acetate buffer (pH 4.7), add 4.7 μL of 21.6 mM peptide reagent (30 equivalents to the antibody), and warm to room temperature. and stirred for 1 hour. The reaction solution was added to a NAP-5 column to stop the reaction and replaced with 20 mM PBS buffer. When the mass was measured by MALDI-TOFMS, no peak derived from the raw material trastuzumab was confirmed, and a peak at 152639 was observed for a product into which one binding peptide had been introduced.

(16-5) HIC-UPLC Analysis of Specific Modification of Anti-HER2 IgG Antibody Trastuzumab The antibody/peptide complex produced in (16-4) and the starting antibody were analyzed by HIC-HPLC. A Butyl-NPR (TOSOH) 4.6×350 mm 2.5 μm column was used. A_Buffer _: 0.1M PiNa, 2.3M ( _NH4 ) _2SO4 , p
H7.0, B_Buffer: 0.1 M PiNa, pH 7.0, flow rate 1.0 ml/min, gradient B 0→100%, 12 min (data collection 20 min), column temperature 25° C., thermostat temperature RT, A detector was used for detection at two wavelengths of UV 225 nm (Ch1) and 280 nm (Ch2).

The samples were reacted under the following conditions.
a: Trastuzumab raw material b: Trastuzumab + peptide disulfide linker conjugate-NHS activated form 30 equivalents;

In the case of UV 225 nm, Retension Time 9.454 minutes is considered to be a trastuzumab raw material, 9.845 minutes is a compound in which one peptide is introduced into trastuzumab, and 10.173 minutes is a compound in which two peptides are introduced into trastuzumab. . In the case of UV 280 nm, Retension Time 9.541 minutes is considered to be a trastuzumab raw material, 9.936 minutes is a compound in which one peptide is introduced into trastuzumab, and 10.254 minutes is a compound in which two peptides are introduced into trastuzumab. (Fig. 27).

[Example 17: Synthesis of a compound (peptide disulfide linker conjugate-NHS activator) having an affinity substance for soluble proteins, a cleavable moiety and a reactive group, and modification of the anti-HER2 IgG antibody trastuzumab using the compound and its analysis]
(17-1) Synthesis of IgG1 Fc-Binding Peptide The sequence of Ac-RGNCAWHRGKLVWCTYH-NH ₂ (SEQ ID NO: 43) was synthesized by the Fmoc solid-phase synthesis method. Liberty Blue manufactured by CEM was used as a peptide synthesizer. All reagents used are manufactured by Watanabe Chemical Industry. Resin was Fmoc-NH-SAL-PEG Resin HL, arginine (R), cysteine (C) and histidine (H) were double-coupled. Cutting from Resin was performed in a solution of trifluoroacetic acid:water:triisopropylsilane:ethanedithiol=94:2.5:1.0:2.5 under stirring conditions for 3 hours. After cutting out, the resin was removed by filtration, and the trifluoroacetic acid was removed. Diethyl ether was added to the resulting crystals for ether precipitation, and the resulting white crystals were collected by filtration. This was dissolved in an aqueous 0.05% trifluoroacetic acid solution and subjected to reversed-phase high performance liquid chromatography using octadodecyl chemically bonded silica gel as a packing material. Elution was performed with the mixed solution, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain the desired product (64.8 mg, 30.5 μmol).

MS (ESI) m/z: z=3 710.45 [M+3H] ³⁺

上記アミノ酸配列は、配列番号４３である。 (17-2) Formation of intramolecular disulfide bond at Cys at positions 4 and 14 of Ac-RGNCAWHRGKLVWCTYH-NH ₂ (SEQ ID NO: 43)

The amino acid sequence is SEQ ID NO:43.

The peptide (64.8 mg, 30.5 µmol) synthesized in (17-2) was dissolved in DMSO (5.00 mL), 2M NH ₃ -MeOH (31.0 µL, 61.0 µmol), hydrogen peroxide solution (62 .0 μL, 610 μmol) was added and stirred at room temperature for 20 hours. A 2M trifluoroacetic acid aqueous solution was added to the reaction solution to stop the reaction, and the solution was dissolved in a 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. , eluted with a mixed solution of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain the target product (35.3 mg, 16.6 μmol).

MS (ESI) m/z: z=3 709.75 [M+3H] ^<3+> , z=4 532.55 [M+4H] ^<4+>

上記アミノ酸配列は、配列番号４３である。 (17-3) Linking disulfide linker and peptide

The amino acid sequence is SEQ ID NO:43.

Ac-RGNCAWHRGKLVWCTYH-NH ₂ (SEQ ID NO: 43) (35.3 mg, 16.6 μmol) synthesized in (17-2), provided that two cysteines at positions 4 and 14 each form an intramolecular disulfide bond ) was dissolved in N,N-dimethylformamide (1.00 mL), and di(N-succinimidyl) 3,3′-dithiodipropionate (269 mg, 0.664 mmol) was dissolved in acetonitrile (2.00 mL). was added and stirred at room temperature for 24 hours. After removing acetonitrile by concentrating under reduced pressure, this was dissolved in 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. Elution was performed with a mixed solution of water and acetonitrile containing 0.05% acetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain the peptide disulfide linker conjugate-NHS activated product (17.5 mg, 7.25 μmol). .

MS (ESI) m/z: z=3 806.20 [M+3H] ^<3+> , z=4 604.90 [M+4H] ^<4+>
HPLC Purity: 100%

(17-4) Specific modification of anti-HER2 IgG antibody trastuzumab and analysis by ESI-TOFMS The peptide disulfide linker conjugate-NHS-activated product synthesized in (17-3) was dissolved in N,N'-dimethylformamide. 6 mM. Dissolve 500 μg of anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical Co., Ltd.) in 46.9 μL of 60 mM sodium acetate buffer (pH 4.7), add 4.7 μL of 21.6 mM peptide reagent (30 equivalents to the antibody), and warm to room temperature. and stirred for 1 hour. The reaction solution was added to a NAP-5 column to stop the reaction and replaced with 20 mM PBS buffer. When the mass was measured by ESI-TOFMS, a peak was observed at 148235 for the raw material trastuzumab, and no peak derived from the product into which the binding peptide had been introduced was observed.

(17-5) Confirmation of heavy chain selectivity by ESI-TOFMS analysis under reducing conditions of a specific modification of trastuzumab 7 mM tris(2-carboxyethyl)phosphine on the antibody/peptide complex produced in (17-4) 5 μL of hydrochloride solution (equal volume to antibody) was added and stirred at room temperature for 20 minutes. When the mass was measured by ESI-TOFMS, light chain peaks were observed at 50594 and 50753 where a thiopropionyl group was introduced into the heavy chain of the product and at 23435 which was the same as the raw material.

(17-6) HIC-HPLC Analysis of Specific Modification of Anti-HER2 IgG Antibody Trastuzumab The antibody/peptide complex produced in (17-4) and the starting antibody were analyzed by HIC. A Butyl-NPR (TOSOH) 4.6×350 mm 2.5 μm column was used. A_Buffer: 0.1 M PiNa, 2.3 M (NH ₄ ) ₂ SO ₄ , pH 7.0, B_Buffer: 0.1 M PiNa, pH 7.0, flow rate 1.0 ml/min, gradient B 0→100%, Detection was performed for 12 minutes (data collection: 20 minutes), the column temperature was 25° C., the thermostat temperature was RT, and the detector was UV 225 nm (Ch1) and 280 nm (Ch2).

The samples were reacted under the following conditions.
a: Trastuzumab raw material b: Trastuzumab + peptide disulfide linker conjugate-NHS activated form 30 equivalents

In the case of UV 225 nm, Retension Time 9.469 minutes is considered to be the trastuzumab raw material, and 10.180 minutes is the peak derived from the peptide. In the case of UV 280 nm, Retension Time 9.563 minutes is considered to be the trastuzumab raw material and 10.270 minutes is the peak derived from the peptide (Fig. 28).

[Example 18: Synthesis of a compound (peptide disulfide linker conjugate-NHS activator) having an affinity substance for soluble proteins, a cleavable moiety and a reactive group, and modification of the anti-HER2 IgG antibody trastuzumab using the compound and its analysis]
(18-1) Synthesis of IgG1 Fc-Binding Peptide The sequence of Ac-RGNCKWHRGELVWCTYH-NH ₂ (SEQ ID NO: 44) was synthesized by the Fmoc solid-phase synthesis method. Liberty Blue manufactured by CEM was used as a peptide synthesizer. All reagents used are manufactured by Watanabe Chemical Industry. Resin was Fmoc-NH-SAL-PEG Resin HL, arginine (R), cysteine (C) and histidine (H) were double-coupled. Cutting from Resin was performed in a solution of trifluoroacetic acid:water:triisopropylsilane:ethanedithiol=94:2.5:1.0:2.5 under stirring conditions for 3 hours. After cutting out, the resin was removed by filtration, and the trifluoroacetic acid was removed. Diethyl ether was added to the resulting crystals for ether precipitation, and the resulting white crystals were collected by filtration. This was dissolved in an aqueous 0.05% trifluoroacetic acid solution and subjected to reversed-phase high performance liquid chromatography using octadodecyl chemically bonded silica gel as a packing material. Elution was performed with the mixed solution, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain the desired product (77.8 mg, 35.6 μmol).

MS (ESI) m/z: z = 3 729.70 [M+H] ⁺ , z = 4 547.60 [M+H] ⁺

上記アミノ酸配列は、配列番号４４である。 (18-2) Formation of intramolecular disulfide bond at Cys at positions 4 and 14 of Ac-RGNCKWHRGELVWCTYH-NH ₂ (SEQ ID NO: 44)

The amino acid sequence is SEQ ID NO:44.

The peptide (77.8 mg, 35.6 µmol) synthesized in (18-1) was dissolved in DMSO (5.00 mL), 2M NH ₃ -MeOH (71.0 µL, 142 µmol), hydrogen peroxide solution (145 µL, 1 .42 mmol) was added and stirred at room temperature for 20 hours. A 2M trifluoroacetic acid aqueous solution was added to the reaction solution to stop the reaction, and the solution was dissolved in a 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. , eluted with a mixed solution of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain the target product (40.0 mg, 18.3 μmol).

MS (ESI) m/z: z=3 729.00 [M+3H] ^<3+> , z=4 547.05 [M+4H] ^<4+>

上記アミノ酸配列は、配列番号４４である。 (18-3) Linking disulfide linker and peptide

The amino acid sequence is SEQ ID NO:44.

Ac-RGNCKWHRGELVWCTYH-NH ₂ (SEQ ID NO: 44) synthesized in (18-2) (40.0 mg, 18.3 μmol, provided that two cysteines at positions 4 and 14 each form an intramolecular disulfide bond ) was dissolved in N,N-dimethylformamide (1.00 mL), and di(N-succinimidyl) 3,3′-dithiodipropionate (296 mg, 0.733 mmol) was dissolved in acetonitrile (2.00 mL). was added and stirred at room temperature for 24 hours. After removing acetonitrile by concentrating under reduced pressure, this was dissolved in 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. Elution was performed with a mixed solvent of water and acetonitrile containing 0.05% acetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain the peptide disulfide linker conjugate-NHS activated product (20.0 mg, 8.09 μmol). .

MS (ESI) m/z: z=3 825.55 [M+3H] ^<3+> , z=4 619.45 [M+4H] ^<4+>
HPLC Purity: 31%

(18-4) Specific modification of anti-HER2 IgG antibody trastuzumab and analysis by ESI-TOFMS The peptide disulfide linker conjugate-NHS-activated product synthesized in (18-3) was dissolved in N,N'-dimethylformamide. 6 mM. Dissolve 500 μg of anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical Co., Ltd.) in 46.9 μL of 60 mM sodium acetate buffer (pH 4.7), add 4.7 μL of 21.6 mM peptide reagent (30 equivalents to the antibody), and warm to room temperature. and stirred for 1 hour. The reaction solution was added to a NAP-5 column to stop the reaction and replaced with 20 mM PBS buffer. When the mass was measured by ESI-TOFMS, a peak was observed at 148222 for the starting trastuzumab and a peak at 150583 for the product into which one binding peptide was introduced.

(18-5) Confirmation of heavy chain selectivity by ESI-TOFMS analysis under reducing conditions of a specific modification of trastuzumab 7 mM tris(2-carboxyethyl)phosphine on the antibody/peptide complex produced in (18-4) 5 μL of hydrochloride solution (equal volume to antibody) was added and stirred at room temperature for 20 minutes. When the mass was measured by ESI-TOFMS, light chain peaks were observed at 50680 and 50719 where a thiopropionyl group was introduced into the heavy chain of the product and at 23438 which was the same as the raw material.

(18-6) HIC-HPLC Analysis of Specific Modification of Anti-HER2 IgG Antibody Trastuzumab The antibody/peptide complex produced in (18-4) and the starting antibody were analyzed by HIC-HPLC. A Butyl-NPR (TOSOH) 4.6×350 mm 2.5 μm column was used. A_Buffer: 0.1 M PiNa, 2.3 M (NH ₄ ) ₂ SO ₄ , pH 7.0, B_Buffer: 0.1 M PiNa, pH 7.0, flow rate 1.0 ml/min, gradient B 0→100%, Detection was performed for 12 minutes (data collection: 20 minutes), the column temperature was 25° C., the thermostat temperature was RT, and the detector was UV 225 nm (Ch1) and 280 nm (Ch2).

The samples were reacted under the following conditions.
a: Trastuzumab raw material b: Trastuzumab + peptide disulfide linker conjugate-NHS activated form 30 equivalents;

In the case of UV225 nm, Retension Time 9.462 minutes is considered to be the trastuzumab raw material, and 10.142 minutes is considered to be a compound in which one peptide is introduced into trastuzumab. In the case of UV 280 nm, Retension Time 9.554 minutes is considered to be the trastuzumab raw material, and 10.230 minutes is considered to be a compound in which one peptide is introduced into trastuzumab (Fig. 29).

[Example 19: Synthesis of a compound (peptide disulfide linker conjugate-NHS activator) having an affinity substance for soluble proteins, a cleavable moiety and a reactive group, and modification of the anti-HER2 IgG antibody trastuzumab using the compound and its analysis]
(19-1) Synthesis of IgG1 Fc-Binding Peptide The sequence of Ac-RGNCKWHRGQLVWCTYH-NH ₂ (SEQ ID NO: 45) was synthesized by the Fmoc solid-phase synthesis method. Liberty Blue manufactured by CEM was used as a peptide synthesizer. All reagents used are manufactured by Watanabe Chemical Industry. Resin was Fmoc-NH-SAL-PEG Resin HL, arginine (R), cysteine (C) and histidine (H) were double-coupled. Cutting from Resin was performed in a solution of trifluoroacetic acid:water:triisopropylsilane:ethanedithiol=94:2.5:1.0:2.5 under stirring conditions for 3 hours. After cutting out, the resin was removed by filtration, and the trifluoroacetic acid was removed. Diethyl ether was added to the resulting crystals for ether precipitation, and the resulting white crystals were collected by filtration. This was dissolved in an aqueous 0.05% trifluoroacetic acid solution and subjected to reversed-phase high performance liquid chromatography using octadodecyl chemically bonded silica gel as a packing material. Elution was performed with the mixed solution, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain the desired product (74.1 mg, 34.0 μmol).

MS (ESI) m/z: z=3 729.40 [M+3H] ^<3+> , z=4 547.35 [M+4H] ^<4+>

上記アミノ酸配列は、配列番号４５である。 (19-2) Formation of intramolecular disulfide bond at Cys at positions 4 and 14 of Ac-RGNCKWHRGQLVWCTYH-NH ₂ (SEQ ID NO: 45)

The amino acid sequence is SEQ ID NO:45.

The peptide (74.1 mg, 34.0 μmol) synthesized in (19-2) was dissolved in DMSO (5.00 mL), 2M NH ₃ -MeOH (67.8 μL, 0.136 mmol), and hydrogen peroxide solution (139 μL). , 1.36 mmol) was added and stirred at room temperature for 20 hours. A 2M trifluoroacetic acid aqueous solution was added to the reaction solution to stop the reaction, and the solution was dissolved in a 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. , eluted with a mixed solution of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain the desired product (38.2 mg, 17.5 μmol).

MS (ESI) m/z: z=3 728.80 [M+3H] ^<3+> , z=4 546.85 [M+4H] ^<4+>

上記アミノ酸配列は、配列番号４５である。 (19-3) Linking disulfide linker and peptide

The amino acid sequence is SEQ ID NO:45.

Ac-RGNCKWHRGQLVWCTYH-NH ₂ (SEQ ID NO: 45) synthesized in (19-2) (38.2 mg, 17.5 μmol, provided that two cysteines at positions 4 and 14 each form an intramolecular disulfide bond ) was dissolved in N,N-dimethylformamide (1.00 mL), and di(N-succinimidyl) 3,3′-dithiodipropionate (283 mg, 0.70 mmol) was dissolved in acetonitrile (2.00 mL). was added and stirred at room temperature for 24 hours. After removing acetonitrile by concentrating under reduced pressure, the residue was dissolved in 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material to remove trifluoroacetic acid. Elution was performed with a mixed solution of water and acetonitrile containing 0.05%, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain the peptide disulfide linker conjugate-NHS activated product (16.5 mg, 6.68 μmol). .

MS (ESI) m/z: z=3 825.25 [M+3H] ^<3+> , z=4 619.25 [M+4H] ^<4+>
HPLC Purity: 100%

(19-4) Specific modification of anti-HER2 IgG antibody trastuzumab and analysis by ESI-TOFMS The peptide disulfide linker conjugate-NHS-activated product synthesized in (19-3) was dissolved in N,N'-dimethylformamide. 6 mM. Dissolve 500 μg of anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical Co., Ltd.) in 46.9 μL of 60 mM sodium acetate buffer (pH 4.7), add 4.7 μL of 21.6 mM peptide reagent (30 equivalents to the antibody), and warm to room temperature. and stirred for 1 hour. The reaction solution was added to a NAP-5 column to stop the reaction and replaced with 20 mM PBS buffer. When the mass was measured by ESI-TOFMS, a peak was observed at 148064 for the raw material trastuzumab and a peak at 150600 for the product into which one binding peptide was introduced.

(19-5) Confirmation of heavy chain selectivity by ESI-TOFMS analysis under reducing conditions of a specific modification of trastuzumab 7 mM tris(2-carboxyethyl)phosphine on the antibody/peptide complex produced in (19-4) 5 μL of hydrochloride solution (equal volume to antibody) was added and stirred at room temperature for 20 minutes. When the mass was measured by ESI-TOFMS, light chain peaks were observed at 50,683 and 50,717 in which a thiopropionyl group was introduced into the heavy chain, and at 23,440, which was the same as the starting material.

(19-5) HIC-HPLC Analysis of Specific Modification of Anti-HER2 IgG Antibody Trastuzumab The antibody/peptide complex produced in (19-4) and the starting antibody were analyzed by HIC-HPLC. A Butyl-NPR (TOSOH) 4.6×350 mm 2.5 μm column was used. A_Buffer: 0.1 M PiNa, 2.3 M (NH ₄ ) ₂ SO ₄ , pH 7.0, B_Buffer: 0.1 M PiNa, pH 7.0, flow rate 1.0 ml/min, gradient B 0→100%, Detection was performed for 12 minutes (data collection: 20 minutes), the column temperature was 25° C., the thermostat temperature was RT, and the detector was UV 225 nm (Ch1) and 280 nm (Ch2).

The samples were reacted under the following conditions.
a: Trastuzumab raw material b: Trastuzumab + peptide disulfide linker conjugate-NHS activated form 30 equivalents;

In the case of UV225 nm, Retension Time 9.458 minutes is considered to be the trastuzumab raw material, and 10.137 minutes is considered to be a compound in which one peptide is introduced into trastuzumab. In the case of UV 280 nm, Retension Time 9.550 minutes is considered to be the trastuzumab raw material, and 10.225 minutes is considered to be a compound in which one peptide is introduced into trastuzumab (Fig. 30).

[Example 20: Synthesis of a compound (peptide disulfide linker conjugate-NHS activator) having an affinity substance for soluble proteins, a cleavable moiety and a reactive group, and modification of the anti-HER2 IgG antibody trastuzumab using the compound and its analysis]
(20-1) Synthesis of IgG1 Fc-Binding Peptide The sequence of Ac-RGNCKYHLGELVWCTYH-NH ₂ (SEQ ID NO: 46) was synthesized by Fmoc solid-phase synthesis. Liberty Blue manufactured by CEM was used as a peptide synthesizer. All reagents used are manufactured by Watanabe Chemical Industry. Resin was Fmoc-NH-SAL-PEG Resin HL, arginine (R), cysteine (C) and histidine (H) were double-coupled. Cutting from Resin was performed in a solution of trifluoroacetic acid:water:triisopropylsilane:ethanedithiol=94:2.5:1.0:2.5 under stirring conditions for 3 hours. After cutting out, the resin was removed by filtration, and the trifluoroacetic acid was removed. Diethyl ether was added to the resulting crystals for ether precipitation, and the resulting white crystals were collected by filtration. This was dissolved in an aqueous 0.05% trifluoroacetic acid solution and subjected to reversed-phase high performance liquid chromatography using octadodecyl chemically bonded silica gel as a packing material. Elution was performed with the mixed solution, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain the desired product (34.5 mg, 16.3 μmol).

MS (ESI) m/z: z=3 707.80 [M+3H] ^<3+> , z=4 531.10 [M+4H] ^<4+>

上記アミノ酸配列は、配列番号４６である。 (20-2) Formation of intramolecular disulfide bond at Cys at positions 4 and 14 of Ac-RGNCKYHLGELVWCTYH-NH ₂ (SEQ ID NO: 46)

The amino acid sequence is SEQ ID NO:46.

The peptide synthesized in (20-1) (34.5 mg, 16.3 μmol) was dissolved in DMSO (5.00 mL), 2M NH ₃ -MeOH (17.0 μL, 32.6 μmol), hydrogen peroxide solution (33 .0 μL, 0.326 mmol) was added and stirred at room temperature for 20 hours. A 2M trifluoroacetic acid aqueous solution was added to the reaction solution to stop the reaction, and the solution was dissolved in a 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. , eluted with a mixed solution of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain the target product (15.6 mg, 7.37 μmol).

MS (ESI) m/z: z=3 707.10 [M+3H] ^<3+> , z=4 530.60 [M+4H] ^<4+>

上記アミノ酸配列は、配列番号４６である。 (20-3) Linking disulfide linker and peptide

The amino acid sequence is SEQ ID NO:46.

Ac-RGNCKYHLGELVWCTYH-NH ₂ (SEQ ID NO: 46) synthesized in (20-2) (15.6 mg, 7.37 μmol, provided that two cysteines at positions 4 and 14 each form an intramolecular disulfide bond) ) was dissolved in N,N-dimethylformamide (1.00 mL), and di(N-succinimidyl) 3,3′-dithiodipropionate (119 mg, 0.295 mmol) was dissolved in acetonitrile (2.00 mL). was added and stirred at room temperature for 24 hours. After removing acetonitrile by concentrating under reduced pressure, this was dissolved in 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. Elution was performed with a mixed solution of water and acetonitrile containing 0.05% acetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain the peptide disulfide linker conjugate-NHS activated product (5.00 mg, 2.08 μmol). .

MS (ESI) m/z: z=2 1204.85 [M+2H] ^<2+> , z=3 803.50 [M+3H] ^<3+>
HPLC Purity: 70%

(20-4) Specific modification of anti-HER2 IgG antibody trastuzumab and analysis by ESI-TOFMS The peptide disulfide linker conjugate-NHS-activated product synthesized in (20-3) was dissolved in N,N'-dimethylformamide. 6 mM. Dissolve 500 μg of anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical Co., Ltd.) in 46.9 μL of 60 mM sodium acetate buffer (pH 4.7), add 4.7 μL of 21.6 mM peptide reagent (30 equivalents to the antibody), and warm to room temperature. and stirred for 1 hour. The reaction solution was added to a NAP-5 column to stop the reaction and replaced with 20 mM PBS buffer.

(20-5) HIC-HPLC Analysis of Specific Modification of Anti-HER2 IgG Antibody Trastuzumab The antibody/peptide complex produced in (20-4) and the starting antibody were analyzed by HIC-HPLC. A Butyl-NPR (TOSOH) 4.6×350 mm 2.5 μm column was used. A_Buffer: 0.1 M PiNa, 2.3 M (NH ₄ ) ₂ SO ₄ , pH 7.0, B_Buffer: 0.1 M PiNa, pH 7.0, flow rate 1.0 ml/min, gradient B 0→100%, Detection was performed for 12 minutes (data collection: 20 minutes), the column temperature was 25° C., the thermostat temperature was RT, and the detector was UV 225 nm (Ch1) and 280 nm (Ch2).

The samples were reacted under the following conditions.
a: Trastuzumab raw material b: Trastuzumab + peptide disulfide linker conjugate-NHS activated form 30 equivalents;

In the case of UV 225 nm, a peak of the trastuzumab raw material was observed at Retension Time 9.581 minutes, and a peak was observed at 10.089 minutes. In the case of UV 280 nm, a peak of the trastuzumab raw material was observed at a Retension Time of 9.663 minutes, and a peak was observed at 10.307 minutes (Fig. 31).

[Example 21: Synthesis of a compound (peptide disulfide linker conjugate-NHS activator) having an affinity substance for soluble proteins, a cleavable moiety and a reactive group, and modification of the anti-HER2 IgG antibody trastuzumab using the compound and its analysis]
(21-1) Synthesis of IgG1 Fc-Binding Peptide The sequence of Ac-RGNCKYHLGQLVWCTYH-NH ₂ (SEQ ID NO: 47) was synthesized by the Fmoc solid-phase synthesis method. Liberty Blue manufactured by CEM was used as a peptide synthesizer. All reagents used are manufactured by Watanabe Chemical Industry. Resin was Fmoc-NH-SAL-PEG Resin HL, arginine (R), cysteine (C) and histidine (H) were double-coupled. Cutting from Resin was performed in a solution of trifluoroacetic acid:water:triisopropylsilane:ethanedithiol=94:2.5:1.0:2.5 under stirring conditions for 3 hours. After cutting out, the resin was removed by filtration, and the trifluoroacetic acid was removed. Diethyl ether was added to the resulting crystals for ether precipitation, and the resulting white crystals were collected by filtration. This was dissolved in an aqueous 0.05% trifluoroacetic acid solution and subjected to reversed-phase high performance liquid chromatography using octadodecyl chemically bonded silica gel as a packing material. Elution was performed with the mixed solution, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain the desired product (53.0 mg, 25.0 μmol).

MS (ESI) m/z: z=3 707.40 [M+3H] ^<3+> , z=4 530.80 [M+4H] ^<4+>

上記アミノ酸配列は、配列番号４７である。 (21-2) Formation of intramolecular disulfide bond at Cys at positions 4 and 14 of Ac-RGNCKYHLGQLVWCTYH-NH ₂ (SEQ ID NO: 47)

The amino acid sequence is SEQ ID NO:47.

The peptide (53.0 mg, 25.0 μmol) synthesized in (21-1) was dissolved in DMSO (5.00 mL), 2M NH ₃ -MeOH (25.0 μL, 50.0 μmol),
A hydrogen peroxide solution (51.0 μL, 0.500 mmol) was added, and the mixture was stirred at room temperature for 20 hours. A 2M trifluoroacetic acid aqueous solution was added to the reaction solution to stop the reaction, and the solution was dissolved in a 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. , eluted with a mixed solution of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain the desired product (23.2 mg, 10.9 μmol).

MS (ESI) m/z: z=3 706.75 [M+3H] ^<3+> , z=4 530.35 [M+4H] ^<4+>

上記アミノ酸配列は、配列番号４７である。 (21-3) Linking disulfide linker and peptide

The amino acid sequence is SEQ ID NO:47.

Ac-RGNCKYHLGQLVWCTYH-NH ₂ (SEQ ID NO: 47) (23.2 mg, 10.9 μmol) synthesized in (21-2), provided that two cysteines at positions 4 and 14 each form an intramolecular disulfide bond ) was dissolved in N,N-dimethylformamide (1.00 mL), and di(N-succinimidyl) 3,3′-dithiodipropionate (176 mg, 0.436 mmol) was dissolved in acetonitrile (2.00 mL). was added and stirred at room temperature for 24 hours. After removing acetonitrile by concentrating under reduced pressure, this was dissolved in 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. Elution was performed with a mixed solution of water and acetonitrile containing 0.05% acetic acid, and each fraction was confirmed by LC-MS. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain the peptide disulfide linker conjugate-NHS activated product (6.40 mg, 2.66 μmol). rice field.

MS (ESI) m/z: z=2 1204.30 [M+2H] ^<2+> , z=3 803.15 [M+3H] ^<3+>
HPLC Purity: 61%

(21-4) Specific modification of anti-HER2 IgG antibody trastuzumab and analysis by ESI-TOFMS The peptide disulfide linker conjugate-NHS-activated product synthesized in (21-3) was dissolved in N,N'-dimethylformamide. 6 mM. Dissolve 500 μg of anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical Co., Ltd.) in 46.9 μL of 60 mM sodium acetate buffer (pH 4.7), add 4.7 μL of 21.6 mM peptide reagent (30 equivalents to the antibody), and warm to room temperature. and stirred for 1 hour. The reaction solution was added to a NAP-5 column to stop the reaction and replaced with 20 mM PBS buffer. When the mass was measured by ESI-TOFMS, a peak was observed at 148069 for the raw material trastuzumab, a peak was observed at 150515 for the product into which one binding peptide was introduced, and two binding peptides were introduced. A peak was observed at 152,972.

(21-5) Confirmation of heavy chain selectivity by ESI-TOFMS analysis under reducing conditions of a specific modification of trastuzumab 7 mM tris(2-carboxyethyl)phosphine on the antibody/peptide complex produced in (21-4) 5 μL of hydrochloride solution (equal volume to antibody) was added and stirred at room temperature for 20 minutes. When the mass was measured by ESI-TOFMS, light chain peaks were observed at 50683 and 50844 where a thiopropionyl group was introduced into the heavy chain of the product, and at 23439 which was the same as the raw material.

(21-6) HIC-HPLC Analysis of Specific Modification of Anti-HER2 IgG Antibody Trastuzumab The antibody/peptide complex produced in (21-4) and the starting antibody were analyzed by HIC-HPLC. A Butyl-NPR (TOSOH) 4.6×350 mm 2.5 μm column was used. A_Buffer: 0.1 M PiNa, 2.3 M (NH ₄ ) ₂ SO ₄ , pH 7.0, B_Buffer: 0.1 M PiNa, pH 7.0, flow rate 1.0 ml/min, gradient B 0→100%, Detection was performed for 12 minutes (data collection: 20 minutes), the column temperature was 25° C., the thermostat temperature was RT, and the detector was UV 225 nm (Ch1) and 280 nm (Ch2).

The samples were reacted under the following conditions.
a: Trastuzumab raw material b: Trastuzumab + peptide disulfide linker conjugate-NHS activated form 30 equivalents;

In the case of UV225 nm, the retention time of 9.454 minutes is considered to be the trastuzumab raw material, 9.877 minutes is considered to be the compound in which one peptide was introduced into trastuzumab, and 10.230 minutes is considered to be the compound into which two peptides were introduced. In the case of UV 280 nm, Retension Time 9.540 minutes is considered to be the trastuzumab raw material, 9.966 minutes is considered to be a compound with one peptide introduced into trastuzumab, and 10.317 minutes is considered to be a compound with two peptides introduced (Fig. 32).

[Example 22: Synthesis of a compound (peptide disulfide linker conjugate-NHS activator) having an affinity substance for soluble proteins, a cleavable moiety and a reactive group, and modification of the anti-HER2 IgG antibody trastuzumab using the compound and its analysis]
(22-1) Synthesis of IgG1 Fc-Binding Peptide The sequence of Ac-DCKWHLGELVWCT-NH ₂ (SEQ ID NO: 48) was synthesized in the same manner as in Example 1 by the Fmoc solid-phase synthesis method. The desired product (71.1 mg, 43.6 μmol) was obtained.
MS (ESI) m/z: z=2 816.350 [M+2H] ²⁺

上記アミノ酸配列は、配列番号４８である。 (22-2) Formation of intramolecular disulfide bond at Cys at positions 2 and 12 of Ac-DCKWHLGELVWCT-NH ₂ (SEQ ID NO: 48)

The amino acid sequence is SEQ ID NO:48.

The peptide (71.1 mg, 43.6 μmol) synthesized in (22-1) was dissolved in DMSO (5.00 mL), 2M NH ₃ -MeOH (43.6 μL, 87.2 μmol), hydrogen peroxide solution (88 .8 μL, 87.2 μmol) was added and stirred at room temperature for 20 hours. A 2M trifluoroacetic acid aqueous solution was added to the reaction solution to stop the reaction, and the solution was dissolved in a 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. , eluted with a mixed solution of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain the above peptide (31.6 mg, 19.4 μmol).

MS (ESI) m/z: z=2 815.350 [M+2H] ²⁺

上記アミノ酸配列は、配列番号４８である。 (22-3) Linking disulfide linker and peptide

The amino acid sequence is SEQ ID NO:48.

Ac-DCKWHLGELVWCT-NH ₂ (SEQ ID NO: 48) synthesized in (22-2) (31.6 mg, 19.4 μmol, provided that the two cysteines at the 2nd and 12th positions each form an intramolecular disulfide bond. ) was dissolved in N,N-dimethylformamide (1.00 mL), and di(N-succinimidyl) 3,3′-dithiodipropionate (312 mg, 776 μmol) was dissolved in acetonitrile (2.00 mL). The mixture was added and stirred at room temperature for 24 hours. After removing acetonitrile by concentrating under reduced pressure, this was dissolved in 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. Elution was performed with a mixed solution of water and acetonitrile containing 0.05% acetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product are collected and concentrated under reduced pressure to remove acetonitrile, followed by lyophilization to give the peptide disulfide linker conjugate-NH
S-activated form (14.2 mg, 7.40 μmol) was obtained.

MS (ESI) m/z: z=2 960.05 [M+2H] ²⁺
HPLC Purity: 70%

(22-4) Specific modification of anti-HER2 IgG antibody trastuzumab and analysis by ESI-TOFMS The peptide disulfide linker conjugate-NHS-activated product synthesized in (22-3) was dissolved in N,N'-dimethylformamide. 6 mM. Dissolve 500 μg of anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical Co., Ltd.) in 46.9 μL of 60 mM sodium acetate buffer (pH 4.7), add 4.7 μL of 21.6 mM peptide reagent (30 equivalents to the antibody), and warm to room temperature. and stirred for 1 hour. The reaction solution was added to a NAP-5 column to stop the reaction and replaced with 20 mM PBS buffer.

(22-5) HIC-HPLC Analysis of Specific Modification of Anti-HER2 IgG Antibody Trastuzumab The antibody/peptide complex produced in (22-4) and the starting antibody were analyzed by HIC-HPLC. A Butyl-NPR (TOSOH) 4.6×350 mm 2.5 μm column was used. A_Buffer: 0.1 M PiNa, 2.3 M (NH ₄ ) ₂ SO ₄ , pH 7.0, B_Buffer: 0.1 M PiNa, pH 7.0, flow rate 1.0 ml/min, gradient B 0→100%, Detection was performed for 12 minutes (data collection: 20 minutes), the column temperature was 25° C., the thermostat temperature was RT, and the detector was UV 225 nm (Ch1) and 280 nm (Ch2).

The samples were reacted under the following conditions.
a: Trastuzumab raw material b: Trastuzumab + peptide disulfide linker conjugate-NHS activated form 30 equivalents;

In the case of UV 225 nm, peaks were observed at Retension Time 11.193 minutes and 11.981 minutes. In the case of UV 280 nm, peaks were observed at Retension Time 11.257 minutes and 12.057 minutes (Fig. 33).

[Example 23: Synthesis of a compound (peptide disulfide linker conjugate-NHS activator) having an affinity substance for soluble proteins, a cleavable moiety and a reactive group, and modification of the anti-HER2 IgG antibody trastuzumab using the compound and its analysis]
(23-1) Synthesis of IgG1 Fc-Binding Peptide The sequence of Ac-DCKYHLGELVWCT-NH ₂ (SEQ ID NO: 49) was synthesized in the same manner as in Example 1 by the Fmoc solid-phase synthesis method. The desired product (28.0 mg, 17.4 μmol) was obtained.

MS (ESI) m/z: z=2 804.80 [M+2H] ²⁺

上記アミノ酸配列は、配列番号４９である。 (23-2) Formation of intramolecular disulfide bond at Cys at positions 2 and 12 of Ac-DCKYHLGELVWCT-NH ₂ (SEQ ID NO: 49)

The amino acid sequence is SEQ ID NO:49.

The peptide (28.0 mg, 17.4 μmol) synthesized in (23-1) was dissolved in DMSO (5.00 mL), 2M NH ₃ -MeOH (17.4 μL, 34.8 μmol), hydrogen peroxide solution (35 .4 μL, 348 μmol) was added and stirred at room temperature for 20 hours. A 2M trifluoroacetic acid aqueous solution was added to the reaction solution to stop the reaction, and the solution was dissolved in a 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. , eluted with a mixed solution of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain the above peptide (18.0 mg, 11.2 μmol).

MS (ESI) m/z: z=2 803.90 [M+2H] ²⁺

上記アミノ酸配列は、配列番号４９である。 (23-3) Linking disulfide linker and peptide

The amino acid sequence is SEQ ID NO:49.

Ac-DCKYHLGELVWCT-NH ₂ (SEQ ID NO: 49) synthesized in (23-2) (18.0 mg, 11.2 μmol, provided that the two cysteines at the 2nd and 12th positions each form an intramolecular disulfide bond. ) was dissolved in N,N-dimethylformamide (1.00 mL), and di(N-succinimidyl) 3,3′-dithiodipropionate (181 mg, 448 μmol) was dissolved in acetonitrile (2.00 mL). The mixture was added and stirred at room temperature for 24 hours. After removing acetonitrile by concentrating under reduced pressure, this was dissolved in 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. Elution was performed with a mixed solution of water and acetonitrile containing 0.05% acetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product are collected and concentrated under reduced pressure to remove acetonitrile, followed by lyophilization to give the peptide disulfide linker conjugate-NH
S-activated form (2.75 mg, 1.45 μmol) was obtained.

MS (ESI) m/z: z=2 948.55 [M+2H] ²⁺
HPLC Purity: 75%

(23-4) Specific modification of anti-HER2 IgG antibody trastuzumab and analysis by ESI-TOFMS The peptide disulfide linker conjugate-NHS-activated product synthesized in (23-3) was dissolved in N,N'-dimethylformamide. 6 mM. Dissolve 500 μg of anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical Co., Ltd.) in 46.9 μL of 60 mM sodium acetate buffer (pH 4.7), add 4.7 μL of 21.6 mM peptide reagent (30 equivalents to the antibody), and warm to room temperature. and stirred for 1 hour. The reaction solution was added to a NAP-5 column to stop the reaction and replaced with 20 mM PBS buffer. When the mass was measured by ESI-TOFMS, a peak was observed at 150001 for the product into which one binding peptide was introduced, and a peak was observed at 151618 for the product into which two binding peptides were introduced.

(23-5) Confirmation of heavy chain selectivity by ESI-TOFMS analysis under reducing conditions of a specific modification of trastuzumab 7 mM tris(2-carboxyethyl)phosphine on the antibody/peptide complex produced in (23-4) 5 μL of hydrochloride solution (equal volume to antibody) was added and stirred at room temperature for 20 minutes. When the mass was measured by ESI-TOFMS, light chain peaks were observed at 50,682 and 50,845, where a thiopropionyl group was introduced into the heavy chain, and at 23,439, which was the same as the starting material.

(23-6) HIC-HPLC Analysis of Specific Modification of Anti-HER2 IgG Antibody Trastuzumab The antibody/peptide complex produced in (23-4) and the starting antibody were analyzed by HIC-HPLC. A Butyl-NPR (TOSOH) 4.6×350 mm 2.5 μm column was used. A_Buffer: 0.1 M PiNa, 2.3 M (NH ₄ ) ₂ SO ₄ , pH 7.0, B_Buffer: 0.1 M PiNa, pH 7.0, flow rate 1.0 ml/min, gradient B 0→100%, Detection was performed for 12 minutes (data collection: 20 minutes), the column temperature was 25° C., the thermostat temperature was RT, and the detector was UV 225 nm (Ch1) and 280 nm (Ch2).

The samples were reacted under the following conditions.
a: Trastuzumab raw material b: Trastuzumab + peptide disulfide linker conjugate-NHS activated form 30 equivalents;

In the case of UV 225 nm, it is considered that the compound with a retention time of 10.347 minutes was a compound in which one peptide was introduced into trastuzumab, and the compound in which two peptides were introduced at 11.015 minutes. In the case of UV 280 nm, the retention time of 10.434 minutes is considered to be a compound in which one peptide was introduced into trastuzumab, and 11.100 minutes was considered to be a compound into which two peptides were introduced (Fig. 34).

[Example 24: Synthesis of a compound (peptide disulfide linker conjugate-NHS activator) having an affinity substance for soluble proteins, a cleavable moiety and a reactive group, and modification of the anti-HER2 IgG antibody trastuzumab using the compound and its analysis]
(24-1) Synthesis of IgG1 Fc-Binding Peptide The sequence of Ac-DCKWHRGELVWCT-NH ₂ (SEQ ID NO: 50) was synthesized in the same manner as in Example 1 by the Fmoc solid-phase synthesis method. The desired product (52.8 mg, 31.5 μmol) was obtained.

MS (ESI) m/z: z=2 837.85 [M+2H] ^<2+> , z=3 558.90 [M+3H] ^<3+>

上記アミノ酸配列は、配列番号５０である。 (24-2) Formation of intramolecular disulfide bond at Cys at positions 2 and 12 of Ac-DCKWHRGELVWCT-NH ₂ (SEQ ID NO: 50)

The amino acid sequence is SEQ ID NO:50.

The peptide (52.8 mg, 31.5 µmol) synthesized in (24-1) was dissolved in DMSO (5.00 mL), 2M NH ₃ -MeOH (31.5 µL, 63.0 µmol), hydrogen peroxide solution (64 .0 μL, 630 μmol) was added and stirred at room temperature for 20 hours. A 2M trifluoroacetic acid aqueous solution was added to the reaction solution to stop the reaction, and the solution was dissolved in a 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. , eluted with a mixed solution of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain the above peptide (26.9 mg, 16.1 μmol).

MS (ESI) m/z: z=2 836.75 [M+2H] ^<2+> , z=3 558.25 [M+3H] ^<3+>

上記アミノ酸配列は、配列番号５０である。 (41-3) Linking disulfide linker and peptide

The amino acid sequence is SEQ ID NO:50.

Ac-DCKWHRGELVWCT-NH ₂ (SEQ ID NO: 50) synthesized in (24-2) (26.9 mg, 16.1 μmol, provided that the two cysteines at positions 2 and 12 each form an intramolecular disulfide bond. ) was dissolved in N,N-dimethylformamide (1.00 mL), and di(N-succinimidyl) 3,3′-dithiodipropionate (260 mg, 644 μmol) was dissolved in acetonitrile (2.00 mL). The mixture was added and stirred at room temperature for 24 hours. After removing acetonitrile by concentrating under reduced pressure, this was dissolved in 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. Elution was performed with a mixed solution of water and acetonitrile containing 0.05% acetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain the peptide disulfide linker conjugate-NHS activated product (8.40 mg, 4.28 μmol). .

MS (ESI) m/z: z=2 981.50 [M+2H] ^<2+> , z=3 654.70 [M+3H] ^<3+>
HPLC Purity: 78%

(24-4) Specific modification of anti-HER2 IgG antibody trastuzumab and analysis by ESI-TOFMS The peptide disulfide linker conjugate-NHS-activated product synthesized in (24-3) was dissolved in N,N'-dimethylformamide. 6 mM. Dissolve 500 μg of anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical Co., Ltd.) in 46.9 μL of 60 mM sodium acetate buffer (pH 4.7), add 4.7 μL of 21.6 mM peptide reagent (30 equivalents to the antibody), and warm to room temperature. and stirred for 1 hour. The reaction solution was added to a NAP-5 column to stop the reaction and replaced with 20 mM PBS buffer. When the mass was measured by ESI-TOFMS, a peak was observed at 150268 for the product into which one binding peptide was introduced, and a peak was observed at 152295 for the product into which two binding peptides were introduced.

(24-5) Confirmation of heavy chain selectivity by ESI-TOFMS analysis under reducing conditions of a specific modification of trastuzumab 7 mM tris(2-carboxyethyl)phosphine on the antibody/peptide complex produced in (24-4) 5 μL of hydrochloride solution (equal volume to antibody) was added and stirred at room temperature for 20 minutes. When the mass was measured by ESI-TOFMS, light chain peaks were observed at 50,682 and 50,841 in which a thiopropionyl group was introduced into the heavy chain, and at 23,438, which was the same as the starting material.

(24-6) HIC-HPLC Analysis of Specific Modification of Anti-HER2 IgG Antibody Trastuzumab The antibody/peptide complex produced in (24-4) and the starting antibody were analyzed by HIC-HPLC. A Butyl-NPR (TOSOH) 4.6×350 mm 2.5 μm column was used. A_Buffer: 0.1 M PiNa, 2.3 M (NH ₄ ) ₂ SO ₄ , pH 7.0, B_Buffer: 0.1 M PiNa, pH 7.0, flow rate 1.0 ml/min, gradient B 0→100%, Detection was performed for 12 minutes (data collection: 20 minutes), the column temperature was 25° C., the thermostat temperature was RT, and the detector was UV 225 nm (Ch1) and 280 nm (Ch2).

The samples were reacted under the following conditions.
a: Trastuzumab raw material b: Trastuzumab + peptide disulfide linker conjugate-NHS activated form 30 equivalents;

In the case of UV225 nm, the compound with a retention time of 10.749 minutes and 11.679 minutes is considered to be a compound in which one peptide was introduced into trastuzumab, and a compound in which two peptides were introduced. In the case of UV 280 nm, it is considered that the compound with a retention time of 10.849 minutes was a compound in which one peptide was introduced into trastuzumab, and the compound in which two peptides were introduced at 11.782 minutes (FIG. 35).

[Example 25: Synthesis of a compound (peptide disulfide linker conjugate-NHS activator) having an affinity substance for soluble proteins, a cleavable moiety and a reactive group, and modification of the anti-HER2 IgG antibody trastuzumab using the compound and its analysis]
(25-1) Synthesis of IgG1 Fc-Binding Peptide The sequence of Ac-DCKWHLGQLVWCT-NH ₂ (SEQ ID NO: 51) was synthesized in the same manner as in Example 1 by the Fmoc solid-phase synthesis method. The desired product (68.5 mg, 42.0 μmol) was obtained.

MS (ESI) m/z: z=2 815.90 [M+2H] ^<2+> , z=3 544.25 [M+3H] ^<3+>

上記アミノ酸配列は、配列番号５１である。 (25-2) Formation of intramolecular disulfide bond at Cys at positions 2 and 12 of Ac-DCKWHLGQLVWCT-NH ₂ (SEQ ID NO: 51)

The amino acid sequence is SEQ ID NO:51.

The peptide (68.5 mg, 42.0 μmol) synthesized in (25-1) was dissolved in DMSO (5.00 mL), 2M NH ₃ -MeOH (42.0 μL, 84.0 μmol), hydrogen peroxide solution (85 .7 μL, 840 μmol) was added and stirred at room temperature for 20 hours. A 2M trifluoroacetic acid aqueous solution was added to the reaction solution to stop the reaction, and the solution was dissolved in a 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. , eluted with a mixed solution of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain the above peptide (30.6 mg, 18.8 μmol).

MS (ESI) m/z: z=2 814.90 [M+2H] ²⁺

上記アミノ酸配列は、配列番号５１である。 (25-3) Linking disulfide linker and peptide

The amino acid sequence is SEQ ID NO:51.

Ac-DCKWHLGQLVWCT-NH ₂ (SEQ ID NO: 51) (30.6 mg, 18.8 μmol) synthesized in (25-2), provided that the two cysteines at the 2nd and 12th positions each form an intramolecular disulfide bond. ) was dissolved in N,N-dimethylformamide (1.00 mL), and di(N-succinimidyl) 3,3′-dithiodipropionate (304 mg, 752 μmol) was dissolved in acetonitrile (2.00 mL). The mixture was added and stirred at room temperature for 24 hours. After removing acetonitrile by concentrating under reduced pressure, this was dissolved in 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. Elution was performed with a mixed solution of water and acetonitrile containing 0.05% acetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain the peptide disulfide linker conjugate-NHS activated product (4.10 mg, 2.14 μmol). .

MS (ESI) m/z: z=2 959.45 [M+2H] ²⁺
HPLC Purity: 74%

(25-4) Specific modification of anti-HER2 IgG antibody trastuzumab and analysis by ESI-TOFMS The peptide disulfide linker conjugate-NHS-activated product synthesized in (42-3) was dissolved in N,N'-dimethylformamide. 6 mM. Dissolve 500 μg of anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical Co., Ltd.) in 46.9 μL of 60 mM sodium acetate buffer (pH 4.7), add 4.7 μL of 21.6 mM peptide reagent (30 equivalents to the antibody), and warm to room temperature. and stirred for 1 hour. The reaction solution was added to a NAP-5 column to stop the reaction and replaced with 20 mM PBS buffer. When the mass was measured by ESI-TOFMS, a peak was observed at 150025 for the product into which one binding peptide was introduced, and a peak was observed at 151878 for the product into which two binding peptides were introduced.

(25-5) Confirmation of heavy chain selectivity by ESI-TOFMS analysis under reducing conditions of a specific modification of trastuzumab 7 mM tris(2-carboxyethyl)phosphine on the antibody/peptide complex produced in (25-4) 5 μL of hydrochloride solution (equal volume to antibody) was added and stirred at room temperature for 20 minutes. When the mass was measured by ESI-TOFMS, light chain peaks were observed at 50682 and 50844 where a thiopropionyl group was introduced into the heavy chain of the product and at 23439 which was the same as the raw material.

(25-6) HIC-HPLC Analysis of Specific Modification of Anti-HER2 IgG Antibody Trastuzumab The antibody/peptide complex produced in (25-4) and the starting antibody were analyzed by HIC-HPLC. A Butyl-NPR (TOSOH) 4.6×350 mm 2.5 μm column was used. A_Buffer: 0.1 M PiNa, 2.3 M (NH ₄ ) ₂ SO ₄ , pH 7.0, B_Buffer: 0.1 M PiNa, pH 7.0, flow rate 1.0 ml/min, gradient B 0→100%, Detection was performed for 12 minutes (data collection: 20 minutes), the column temperature was 25° C., the thermostat temperature was RT, and the detector was UV 225 nm (Ch1) and 280 nm (Ch2).

The samples were reacted under the following conditions.
a: Trastuzumab raw material b: Trastuzumab + peptide disulfide linker conjugate-NHS activated form 30 equivalents;

In the case of UV225 nm, the compound with a retention time of 10.761 minutes and 11.722 minutes is considered to be a compound in which one peptide was introduced into trastuzumab, and a compound in which two peptides were introduced. In the case of UV 280 nm, it is considered that the compound with a retention time of 10.849 minutes was a compound in which one peptide was introduced into trastuzumab, and the compound in which two peptides were introduced at 11.807 minutes (FIG. 36).

[Example 26: Synthesis of a compound (peptide disulfide linker conjugate-NHS activator) having an affinity substance for soluble proteins, a cleavable moiety and a reactive group, and modification of the anti-HER2 IgG antibody trastuzumab using the compound and its analysis]
(26-1) Synthesis of IgG1 Fc-Binding Peptide The sequence of Ac-DCKYHRGELVWCT-NH ₂ (SEQ ID NO: 52) was synthesized in the same manner as in Example 1 by the Fmoc solid-phase synthesis method. The desired product (100 mg, 60.8 μmol) was obtained.

MS (ESI) m/z: z=2 826.35 [M+2H] ^<2+> , z=3 551.25 [M+3H] ^<3+>

上記アミノ酸配列は、配列番号５２である。 (26-2) Formation of intramolecular disulfide bond at Cys at positions 2 and 12 of Ac-DCKYHRGELVWCT-NH ₂ (SEQ ID NO: 52)

The amino acid sequence is SEQ ID NO:52.

The peptide (100 mg, 60.8 μmol) synthesized in (26-1) was dissolved in DMSO (5.00 mL), 2M NH ₃ -MeOH (60.8 μL, 122 μmol), hydrogen peroxide solution (125 μL, 1.22 mmol). ) was added and stirred at room temperature for 20 hours. A 2M trifluoroacetic acid aqueous solution was added to the reaction solution to stop the reaction, and the solution was dissolved in a 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. , eluted with a mixed solution of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain the above peptide (74.5 mg, 45.2 μmol).

MS (ESI) m/z: z=2 825.30 [M+2H] ^<2+> , z=3 550.60 [M+3H] ^<3+>

上記アミノ酸配列は、配列番号５２である。 (26-3) Linking disulfide linker and peptide

The amino acid sequence is SEQ ID NO:52.

Ac-DCKYHRGELVWCT-NH ₂ (SEQ ID NO: 52) (74.5 mg, 45.2 μmol) synthesized in (26-2), provided that the two cysteines at the 2nd and 12th positions each form an intramolecular disulfide bond. ) was dissolved in N,N-dimethylformamide (1.00 mL), and di(N-succinimidyl) 3,3′-dithiodipropionate (731 mg, 1.81 mmol) was dissolved in acetonitrile (2.00 mL). The solution was added and stirred at room temperature for 24 hours. After removing acetonitrile by concentrating under reduced pressure, this was dissolved in 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. Elution was performed with a mixed solution of water and acetonitrile containing 0.05% acetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain the peptide disulfide linker conjugate-NHS activated product (23.0 mg, 18.9 μmol). .

MS (ESI) m/z: z=2 970.00 [M+2H] ²⁺
HPLC Purity: 69%

(26-4) Specific modification of anti-HER2 IgG antibody trastuzumab and analysis by ESI-TOFMS The peptide disulfide linker conjugate-NHS-activated product synthesized in (26-3) was dissolved in N,N'-dimethylformamide. 6 mM. Dissolve 500 μg of anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical Co., Ltd.) in 46.9 μL of 60 mM sodium acetate buffer (pH 4.7), add 4.7 μL of 21.6 mM peptide reagent (30 equivalents to the antibody), and warm to room temperature. and stirred for 1 hour. The reaction solution was added to a NAP-5 column to stop the reaction and replaced with 20 mM PBS buffer. When the mass was measured by ESI-TOFMS, a peak was observed at 147074 for the raw material trastuzumab, and a peak was observed at 150062 for the product into which one binding peptide was introduced. A peak was observed at 151,873. A by-product peak was observed at 154,562.

(26-5) HIC-HPLC Analysis of Specific Modification of Anti-HER2 IgG Antibody Trastuzumab The antibody/peptide complex produced in (26-4) and the starting antibody were analyzed by HIC-HPLC. A Butyl-NPR (TOSOH) 4.6×350 mm 2.5 μm column was used. A_Buffer: 0.1 M PiNa, 2.3 M (NH ₄ ) ₂ SO ₄ , pH 7.0, B_Buffer: 0.1 M PiNa, pH 7.0, flow rate 1.0 ml/min, gradient B 0→100%, Detection was performed for 12 minutes (data collection: 20 minutes), the column temperature was 25° C., the thermostat temperature was RT, and the detector was UV 225 nm (Ch1) and 280 nm (Ch2).

The samples were reacted under the following conditions.
a: Trastuzumab raw material b: Trastuzumab + peptide disulfide linker conjugate-NHS activated form 30 equivalents;

In the case of UV225 nm, the retention time of 9.464 minutes is considered to be the trastuzumab raw material, 10.309 minutes is considered to be the compound in which one peptide is introduced into trastuzumab, and 10.962 minutes is the compound into which two peptides are introduced. In the case of UV 280 nm, the retention time of 9.564 minutes is considered to be the trastuzumab raw material, 10.395 minutes is considered to be a compound with one peptide introduced into trastuzumab, and 11.054 minutes is considered to be a compound with two peptides introduced (Fig. 37).

Example 27: Synthesis of a compound (peptide disulfide linker conjugate-NHS activator) with an affinity for soluble proteins, a cleavable moiety and a reactive group, and modification of the anti-HER2 IgG antibody trastuzumab using the compound and its analysis]
(27-1) Synthesis of IgG1 Fc-Binding Peptide The sequence of Ac-DCKYHLGQLVWCT-NH ₂ (SEQ ID NO: 53) was synthesized in the same manner as in Example 1 by the Fmoc solid-phase synthesis method. The desired product (85.6 mg, 53.3 μmol) was obtained.

MS (ESI) m/z: z=2 804.45 [M+2H] ^<2+> , z=3 536.60 [M+3H] ^<3+>

上記アミノ酸配列は、配列番号５３である。 (27-2) Formation of intramolecular disulfide bond at Cys at positions 2 and 12 of Ac-DCKYHLGQLVWCT-NH ₂ (SEQ ID NO: 53)

The amino acid sequence is SEQ ID NO:53.

The peptide (85.6 mg, 53.3 µmol) synthesized in (27-1) was dissolved in DMSO (5.00 mL), 2M NH ₃ -MeOH (53.5 µL, 107 µmol), hydrogen peroxide solution (109 µL, 1 .07 mmol) was added and stirred at room temperature for 20 hours. A 2M trifluoroacetic acid aqueous solution was added to the reaction solution to stop the reaction, and the solution was dissolved in a 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. , eluted with a mixed solution of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain the above peptide (34.2 mg, 21.3 μmol).

MS (ESI) m/z: z=2 803.40 [M+2H] ²⁺

上記アミノ酸配列は、配列番号５３である。 (27-3) Linking disulfide linker and peptide

The amino acid sequence is SEQ ID NO:53.

Ac-DCKYHLGQLVWCT-NH ₂ (SEQ ID NO: 53) synthesized in (27-2) (34.2 mg, 21.3 μmol, provided that the two cysteines at the 2nd and 12th positions each form an intramolecular disulfide bond. ) was dissolved in N,N-dimethylformamide (1.00 mL), and di(N-succinimidyl) 3,3′-dithiodipropionate (345 mg, 852 μmol) was dissolved in acetonitrile (2.00 mL). The mixture was added and stirred at room temperature for 24 hours. After removing acetonitrile by concentrating under reduced pressure, this was dissolved in 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. Elution was performed with a mixed solution of water and acetonitrile containing 0.05% acetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain the peptide disulfide linker conjugate-NHS activated product (12.0 mg, 6.34 μmol). .

MS (ESI) m/z: z=2 948.00 [M+2H] ²⁺
HPLC Purity: 80%

(27-4) Specific modification of anti-HER2 IgG antibody trastuzumab and analysis by ESI-TOFMS The peptide disulfide linker conjugate-NHS-activated product synthesized in (27-3) was dissolved in N,N'-dimethylformamide. 6 mM. Dissolve 500 μg of anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical Co., Ltd.) in 46.9 μL of 60 mM sodium acetate buffer (pH 4.7), add 4.7 μL of 21.6 mM peptide reagent (30 equivalents to the antibody), and warm to room temperature. and stirred for 1 hour. The reaction solution was added to a NAP-5 column to stop the reaction and replaced with 20 mM PBS buffer. When the mass was measured by ESI-TOFMS, a peak was observed at 147285 for the raw material trastuzumab, and a peak was observed at 150031 for the product into which one binding peptide was introduced. A peak was observed at 151,947. A peak due to a by-product was observed at 154885.

(27-5) Confirmation of heavy chain selectivity by ESI-TOFMS analysis under reducing conditions of a specific modification of trastuzumab 7 mM tris(2-carboxyethyl)phosphine on the antibody/peptide complex produced in (27-4) 5 μL of hydrochloride solution (equal volume to antibody) was added and stirred at room temperature for 20 minutes. When the mass was measured by ESI-TOFMS, peaks were observed at 50685 and 50847 where a thiopropionyl group was introduced into the heavy chain of the product.

(27-6) HIC-HPLC Analysis of Specific Modification of Anti-HER2 IgG Antibody Trastuzumab The antibody/peptide complex produced in (27-4) and the starting antibody were analyzed by HIC-HPLC. A Butyl-NPR (TOSOH) 4.6×350 mm 2.5 μm column was used. A_Buffer: 0.1 M PiNa, 2.3 M (NH ₄ ) ₂ SO ₄ , pH 7.0, B_Buffer: 0.1 M PiNa, pH 7.0, flow rate 1.0 ml/min, gradient B 0→100%, Detection was performed for 12 minutes (data collection: 20 minutes), the column temperature was 25° C., the thermostat temperature was RT, and the detector was UV 225 nm (Ch1) and 280 nm (Ch2).

The samples were reacted under the following conditions.
a: Trastuzumab raw material b: Trastuzumab + peptide disulfide linker conjugate-NHS activated form 30 equivalents;

In the case of UV225 nm, the retention time of 9.468 minutes is considered to be the trastuzumab raw material, 10.360 minutes is considered to be the compound in which one peptide was introduced into trastuzumab, and 11.045 minutes is the compound into which two peptides were introduced. In the case of UV 280 nm, the retention time of 9.575 minutes is considered to be the trastuzumab raw material, 10.449 minutes is considered to be a compound with one peptide introduced into trastuzumab, and 11.137 minutes is considered to be a compound with two peptides introduced (Fig. 38).

[Example 28: Synthesis of a compound (peptide disulfide linker conjugate-NHS activator) having an affinity substance for soluble proteins, a cleavable moiety and a reactive group, and modification of the anti-HER2 IgG antibody trastuzumab using the compound and its analysis]
(28-1) Synthesis of IgG1 Fc-Binding Peptide The sequence of Ac-DCKWHRGQLVWCT-NH ₂ (SEQ ID NO: 54) was synthesized in the same manner as in Example 1 by the Fmoc solid-phase synthesis method. The desired product (89.4 mg, 57.4 μmol) was obtained.

MS (ESI) m/z: z=2 837.30 [M+2H] ^<2+> , z=3 558.60 [M+3H] ^<3+>

上記アミノ酸配列は、配列番号５４である。 (28-2) Formation of intramolecular disulfide bond at Cys at positions 2 and 12 of Ac-DCKWHRGQLVWCT-NH ₂ (SEQ ID NO: 54)

The amino acid sequence is SEQ ID NO:54.

The peptide (89.4 mg, 57.4 μmol) synthesized in (28-1) was dissolved in DMSO (5.00 mL), 2M NH ₃ -MeOH (53.5 μL, 107 μmol), hydrogen peroxide solution (109 μL, 1 .07 mmol) was added and stirred at room temperature for 20 hours. A 2M trifluoroacetic acid aqueous solution was added to the reaction solution to stop the reaction, and the solution was dissolved in a 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. , eluted with a mixed solution of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain the above peptide (64.4 mg, 38.5 μmol).

MS (ESI) m/z: z=2 836.35 [M+2H] ^<2+> , z=3 558.00 [M+3H] ^<3+>

上記アミノ酸配列は、配列番号５４である。 (28-3) Linking disulfide linker and peptide

The amino acid sequence is SEQ ID NO:54.

Ac-DCKWHRGQLVWCT-NH ₂ (SEQ ID NO: 54) (64.4 mg, 38.5 μmol) synthesized in (28-2), provided that the two cysteines at the 2nd and 12th positions each form an intramolecular disulfide bond. ) was dissolved in N,N-dimethylformamide (1.00 mL), and di(N-succinimidyl) 3,3′-dithiodipropionate (623 mg, 1.54 mmol) was dissolved in acetonitrile (2.00 mL). The solution was added and stirred at room temperature for 24 hours. After removing acetonitrile by concentrating under reduced pressure, this was dissolved in 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. Elution was performed with a mixed solution of water and acetonitrile containing 0.05% acetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product are collected and concentrated under reduced pressure to remove acetonitrile, followed by lyophilization to obtain the peptide disulfide linker conjugate-N.
HS activated form (21.8 mg, 11.1 μmol) was obtained.

MS (ESI) m/z: z=2 981.05 [M+2H] ^<2+> , z=3 654.25 [M+3H] ^<3+>
HPLC Purity: 82%

(28-4) Specific modification of anti-HER2 IgG antibody trastuzumab and analysis by ESI-TOFMS The peptide disulfide linker conjugate-NHS-activated product synthesized in (28-3) was dissolved in N,N'-dimethylformamide. 6 mM. Dissolve 500 μg of anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical Co., Ltd.) in 46.9 μL of 60 mM sodium acetate buffer (pH 4.7), add 4.7 μL of 21.6 mM peptide reagent (30 equivalents to the antibody), and warm to room temperature. and stirred for 1 hour. The reaction solution was added to a NAP-5 column to stop the reaction and replaced with 20 mM PBS buffer. When the mass was measured by ESI-TOFMS, a peak was observed at 147548 for the raw material trastuzumab, a peak was observed at 150261 for the product into which one binding peptide was introduced, and two binding peptides were introduced. A peak was observed at 152112. A by-product peak was observed at 154,824.

(28-5) Confirmation of heavy chain selectivity by ESI-TOFMS analysis under reducing conditions of a specific modification of trastuzumab 7 mM tris(2-carboxyethyl)phosphine on the antibody/peptide complex produced in (28-4) 5 μL of hydrochloride solution (equal volume to antibody) was added and stirred at room temperature for 20 minutes. When the mass was measured by ESI-TOFMS, peaks were observed at 50684 and 50845 where a thiopropionyl group was introduced into the heavy chain of the product.

(28-6) HIC-HPLC Analysis of Specific Modification of Anti-HER2 IgG Antibody Trastuzumab The antibody/peptide complex produced in (28-4) and the starting antibody were analyzed by HIC-HPLC. A Butyl-NPR (TOSOH) 4.6×350 mm 2.5 μm column was used. A_Buffer: 0.1 M PiNa, 2.3 M (NH ₄ ) ₂ SO ₄ , pH 7.0, B_Buffer: 0.1 M PiNa, pH 7.0, flow rate 1.0 ml/min, gradient B 0→100%, Detection was performed for 12 minutes (data collection: 20 minutes), the column temperature was 25° C., the thermostat temperature was RT, and the detector was UV 225 nm (Ch1) and 280 nm (Ch2).

The samples were reacted under the following conditions.
a: Trastuzumab raw material b: Trastuzumab + peptide disulfide linker conjugate-NHS activated form 30 equivalents;

In the case of UV225 nm, the retention time of 9.477 minutes is considered to be the trastuzumab raw material, 10.714 minutes is considered to be a compound in which one peptide is introduced into trastuzumab, and 11.650 minutes is considered to be a by-product. In the case of UV 280 nm, Retension Time 9.573 minutes is considered to be the trastuzumab raw material, 10.812 minutes is considered to be a compound in which one peptide is introduced into trastuzumab, and 11.742 minutes is considered to be a by-product (Fig. 39).

[Example 29: Synthesis of a compound (peptide disulfide linker conjugate-NHS activator) having an affinity substance for soluble proteins, a cleavable moiety and a reactive group, and modification of the anti-HER2 IgG antibody trastuzumab using the compound and its analysis]
(47-1) Synthesis of IgG1 Fc-Binding Peptide The sequence of Ac-DCKYHRGQLVWCT-NH ₂ (SEQ ID NO: 55) was synthesized in the same manner as in Example 1 by the Fmoc solid-phase synthesis method. The desired product (93.6 mg, 56.7 μmol) was obtained.

MS (ESI) m/z: z=2 825.80 [M+2H] ^<2+> , z=3 550.90 [M+3H] ^<3+>

上記アミノ酸配列は、配列番号５５である。 (29-2) Formation of intramolecular disulfide bond at Cys at positions 2 and 12 of Ac-DCKYHRGQLVWCT-NH ₂ (SEQ ID NO: 55)

The amino acid sequence is SEQ ID NO:55.

The peptide (93.6 mg, 56.7 μmol) synthesized in (29-1) was dissolved in DMSO (5.00 mL), 2M NH ₃ -MeOH (56.5 μL, 113 μmol), hydrogen peroxide solution (115 μL, 1 .13 mmol) was added and stirred at room temperature for 20 hours. A 2M trifluoroacetic acid aqueous solution was added to the reaction solution to stop the reaction, and the solution was dissolved in a 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. , eluted with a mixed solution of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain the above peptide (71.6 mg, 46.2 μmol).

MS (ESI) m/z: z=2 824.85 [M+2H] ^<2+> , z=3 550.25 [M+3H] ^<3+>

上記アミノ酸配列は、配列番号５５である。 (29-3) Linking disulfide linker and peptide

The amino acid sequence is SEQ ID NO:55.

Ac-DCKYHRGQLVWCT-NH ₂ (SEQ ID NO: 55) synthesized in (29-2)
(71.6 mg, 46.2 μmol, where the two cysteines at the 2nd and 12th positions each form an intramolecular disulfide bond) was dissolved in N,N-dimethylformamide (1.00 mL), A solution of di(N-succinimidyl),3′-dithiodipropionate (747 mg, 1.85 mmol) dissolved in acetonitrile (2.00 mL) was added and stirred at room temperature for 24 hours. After removing acetonitrile by concentrating under reduced pressure, this was dissolved in 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. Elution was performed with a mixed solution of water and acetonitrile containing 0.05% acetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain the peptide disulfide linker conjugate-NHS activated product (25.5 mg, 13.2 μmol). .

MS (ESI) m/z: z=2 969.45 [M+2H] ^<2+> , z=3 646.75 [M+3H] ^<3+>
HPLC Purity: 82%

(29-4) Specific modification of anti-HER2 IgG antibody trastuzumab and analysis by ESI-TOFMS The peptide disulfide linker conjugate-NHS-activated product synthesized in (29-3) was dissolved in N,N'-dimethylformamide. 6 mM. Dissolve 500 μg of anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical Co., Ltd.) in 46.9 μL of 60 mM sodium acetate buffer (pH 4.7), add 4.7 μL of 21.6 mM peptide reagent (30 equivalents to the antibody), and warm to room temperature. and stirred for 1 hour. The reaction solution was added to a NAP-5 column to stop the reaction and replaced with 20 mM PBS buffer. When the mass was measured by ESI-TOFMS, a peak was observed at 149884 for the product into which one binding peptide was introduced, and a peak was observed at 151873 for the product into which two binding peptides were introduced.

(29-5) Confirmation of heavy chain selectivity by ESI-TOFMS analysis under reducing conditions of a specific modification of trastuzumab 7 mM tris(2-carboxyethyl)phosphine on the antibody/peptide complex produced in (29-4) 5 μL of hydrochloride solution (equal volume to antibody) was added and stirred at room temperature for 20 minutes. When the mass was measured by ESI-TOFMS, peaks were observed at 50682 and 50844 where a thiopropionyl group was introduced into the heavy chain of the product.

(29-6) HIC-HPLC Analysis of Specific Modification of Anti-HER2 IgG Antibody Trastuzumab The antibody/peptide complex produced in (29-4) and the starting antibody were analyzed by HIC-HPLC. A Butyl-NPR (TOSOH) 4.6×350 mm 2.5 μm column was used. A_Buffer: 0.1 M PiNa, 2.3 M (NH ₄ ) ₂ SO ₄ , pH 7.0, B_Buffer: 0.1 M PiNa, pH 7.0, flow rate 1.0 ml/min, gradient B 0→100%, Detection was performed for 12 minutes (data collection: 20 minutes), the column temperature was 25° C., the thermostat temperature was RT, and the detector was UV 225 nm (Ch1) and 280 nm (Ch2).

The samples were reacted under the following conditions.
a: Trastuzumab raw material b: Trastuzumab + peptide disulfide linker conjugate-NHS activated form 30 equivalents;

In the case of UV225 nm, the compound with a retention time of 10.292 minutes and 10.949 minutes is considered to be a compound in which one peptide was introduced into trastuzumab, and a compound in which two peptides were introduced. In the case of UV 280 nm, it is considered that the compound with a retention time of 10.381 minutes was a compound in which one peptide was introduced into trastuzumab, and the compound with a retention time of 11.038 minutes was a compound in which two peptides were introduced (FIG. 40).

[Example 30: Synthesis of a compound (peptide disulfide linker conjugate-NHS activator) having an affinity substance for soluble proteins, a cleavable moiety and a reactive group, and modification of the anti-HER2 IgG antibody trastuzumab using the compound and its analysis]
(30-1) Synthesis of IgG1 Fc-Binding Peptide The sequence of Ac-RGNCAWHLGQLVWCKYH-NH ₂ (SEQ ID NO: 56) was synthesized in the same manner as in Example 1 by the Fmoc solid-phase synthesis method. The desired product (54.2 mg, 25.7 μmol) was obtained.

MS (ESI) m/z: z=3 705.05 [M+3H] ^<3+> , z=4 529.10 [M+4H] ^<4+>

上記アミノ酸配列は、配列番号５６である。 (30-2) Formation of intramolecular disulfide bond at Cys at positions 4 and 14 of Ac-RGNCAWHLGQLVWCKYH-NH ₂ (SEQ ID NO: 56)

The amino acid sequence is SEQ ID NO:56.

The peptide (54.2 mg, 25.7 μmol) synthesized in (30-1) was dissolved in DMSO (5.00 mL), 2M NH ₃ -MeOH (25.7 μL, 51.4 μmol), hydrogen peroxide solution (52 .5 μL, 514 μmol) was added and stirred at room temperature for 20 hours. A 2M trifluoroacetic acid aqueous solution was added to the reaction solution to stop the reaction, and the solution was dissolved in a 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. , eluted with a mixed solution of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain the above peptide (13.3 mg, 5.54 μmol).

MS (ESI) m/z: z=3 704.45 [M+3H] ^<3+> , z=4 528.60 [M+4H] ^<4+>

上記アミノ酸配列は、配列番号５６である。 (30-3) Linking disulfide linker and peptide

The amino acid sequence is SEQ ID NO:56.

Ac-RGNCAWHLGQLVWCKYH-NH ₂ (SEQ ID NO: 56) (13.3 mg, 5.54 μmol) synthesized in (30-2), provided that the two cysteines at positions 4 and 14 each form intramolecular disulfide bonds. ) was dissolved in N,N-dimethylformamide (1.00 mL), and di(N-succinimidyl) 3,3′-dithiodipropionate (89.6 mg, 222 μmol) was dissolved in acetonitrile (2.00 mL). The solution was added and stirred at room temperature for 24 hours. After removing acetonitrile by concentrating under reduced pressure, this was dissolved in 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. Elution was performed with a mixed solution of water and acetonitrile containing 0.05% acetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain the peptide disulfide linker conjugate-NHS activated product (7.00 mg, 2.92 μmol). .

MS (ESI) m/z: z=3 800.90 [M+3H] ^<3+> , z=4 600.80 [M+4H] ^<4+>
HPLC Purity: 69%

(30-4) Specific modification of anti-HER2 IgG antibody trastuzumab and analysis by ESI-TOFMS The peptide disulfide linker conjugate-NHS-activated product synthesized in (30-3) was dissolved in N,N'-dimethylformamide. 6 mM. Dissolve 500 μg of anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical Co., Ltd.) in 46.9 μL of 60 mM sodium acetate buffer (pH 4.7), add 4.7 μL of 21.6 mM peptide reagent (30 equivalents to the antibody), and warm to room temperature. and stirred for 1 hour. The reaction solution was added to a NAP-5 column to stop the reaction and replaced with 20 mM PBS buffer. When the mass was measured by ESI-TOFMS, a peak was observed at 148788 for the raw material trastuzumab and a peak at 151375 for the product into which one binding peptide was introduced.

(30-5) Confirmation of heavy chain selectivity by ESI-TOFMS analysis under reducing conditions of a specific modification of trastuzumab 7 mM tris(2-carboxyethyl)phosphine on the antibody/peptide complex produced in (30-4) 5 μL of hydrochloride solution (equal volume to antibody) was added and stirred at room temperature for 20 minutes.
When the mass was measured by ESI-TOFMS, light chain peaks were observed at 50593 and 50756 where a thiopropionyl group was introduced into the heavy chain of the product and at 23337 which was the same as the raw material.

(30-6) HIC-HPLC Analysis of Specific Modification of Anti-HER2 IgG Antibody Trastuzumab The antibody/peptide complex produced in (30-4) and the starting antibody were analyzed by HIC. A Butyl-NPR (TOSOH) 4.6×350 mm 2.5 μm column was used. A_Buffer: 0.1 M PiNa, 2.3 M (NH ₄ ) ₂ SO ₄ , pH 7.0, B_Buffer: 0.1 M PiNa, pH 7.0, flow rate 1.0 ml/min, gradient B 0→100%, Detection was performed for 12 minutes (data collection: 20 minutes), the column temperature was 25° C., the thermostat temperature was RT, and the detector was UV 225 nm (Ch1) and 280 nm (Ch2).

The samples were reacted under the following conditions.
a: Trastuzumab raw material b: Trastuzumab + peptide disulfide linker conjugate-NHS activated form 30 equivalents;

In the case of UV225 nm, Retension Time 9.556 minutes is considered to be the trastuzumab raw material, and 10.259 minutes is considered to be a peak derived from a compound in which one peptide is introduced into trastuzumab. In the case of UV 280 nm, Retension Time 9.549 minutes is considered to be the trastuzumab raw material, and 10.382 minutes is considered to be the peak derived from the compound in which one peptide was introduced into trastuzumab (Fig. 41).

[Example 31: Synthesis of a compound (peptide disulfide linker conjugate-NHS activator) having an affinity substance for soluble proteins, a cleavable moiety and a reactive group, and modification of the anti-HER2 IgG antibody trastuzumab using the compound and its analysis]
(31-1) Synthesis of IgG1 Fc-Binding Peptide The sequence of Ac-RGNCAWHLGELVWCKYH-NH ₂ (SEQ ID NO: 57) was synthesized in the same manner as in Example 1 by the Fmoc solid-phase synthesis method. The desired product (34.0 mg, 16.1 μmol) was obtained.

MS (ESI) m/z: z=3 705.40 [M+3H] ^<3+> , z=4 529.30 [M+4H] ^<4+>

上記アミノ酸配列は、配列番号５７である。 (31-2) Formation of intramolecular disulfide bond at Cys at positions 4 and 14 of Ac-RGNCAWHLGELVWCKYH-NH ₂ (SEQ ID NO: 57)

The amino acid sequence is SEQ ID NO:57.

The peptide (34.0 mg, 16.1 μmol) synthesized in (31-1) was dissolved in DMSO (5.00 mL), 2M NH ₃ -MeOH (16.1 μL, 32.2 μmol), hydrogen peroxide solution (32 .9 μL, 322 μmol) was added and stirred at room temperature for 20 hours. A 2M trifluoroacetic acid aqueous solution was added to the reaction solution to stop the reaction, and the solution was dissolved in a 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. , eluted with a mixed solution of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain the above peptide (13.6 mg, 6.40 μmol).

MS (ESI) m/z: z=3 704.85 [M+3H] ^<3+> , z=4 528.80 [M+4H] ^<4+>

上記アミノ酸配列は、配列番号５７である。 (31-3) Linking disulfide linker and peptide

The amino acid sequence is SEQ ID NO:57.

Ac-RGNCAWHLGELVWCKYH-NH ₂ (SEQ ID NO: 57) synthesized in (31-2) (13.6 mg, 6.40 μmol, provided that two cysteines at positions 4 and 14 each form an intramolecular disulfide bond. ) was dissolved in N,N-dimethylformamide (1.00 mL), and di(N-succinimidyl) 3,3′-dithiodipropionate (104 mg, 256 μmol) was dissolved in acetonitrile (2.00 mL). The mixture was added and stirred at room temperature for 24 hours. After removing acetonitrile by concentrating under reduced pressure, this was dissolved in 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. Elution was performed with a mixed solution of water and acetonitrile containing 0.05% acetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain the peptide disulfide linker conjugate-NHS activated product (1.5 mg, 0.63 μmol). .

MS (ESI) m/z: z=3 801.45 [M+3H] ^<3+> , z=4 601.50 [M+4H] ^<4+>
HPLC Purity: 77%

(31-4) Specific modification of anti-HER2 IgG antibody trastuzumab and analysis by ESI-TOFMS The peptide disulfide linker conjugate-NHS-activated product synthesized in (31-3) was dissolved in N,N'-dimethylformamide. 6 mM. Dissolve 500 μg of anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical Co., Ltd.) in 46.9 μL of 60 mM sodium acetate buffer (pH 4.7), add 4.7 μL of 21.6 mM peptide reagent (30 equivalents to the antibody), and warm to room temperature. and stirred for 1 hour. The reaction solution was added to a NAP-5 column to stop the reaction and replaced with 20 mM PBS buffer. When the mass was measured by ESI-TOFMS, a peak was observed at 148219 for the raw material trastuzumab, and no peak derived from the product into which the binding peptide had been introduced was observed.

(31-5) Confirmation of heavy chain selectivity by ESI-TOFMS analysis under reducing conditions of a specific modification of trastuzumab 7 mM tris(2-carboxyethyl)phosphine on the antibody/peptide complex produced in (31-4) 5 μL of hydrochloride solution (equal volume to antibody) was added and stirred at room temperature for 20 minutes. When the mass was measured by ESI-TOFMS, light chain peaks were observed at 50,592 and 50,762 in which a thiopropionyl group was introduced into the heavy chain, and at 23,338, which was the same as the starting material.

(31-6) HIC-UPLC Analysis of Specific Modification of Anti-HER2 IgG Antibody Trastuzumab The antibody/peptide complex produced in (31-4) and the starting antibody were analyzed by HIC. A Protein-Pak Hi Res HIC Column (Waters) 4.6×100 mm 2.5 μm was used as the column. A_Buffer: 0.1 M PiNa, 2.3 M (NH ₄ ) ₂ SO ₄ , pH 7.0, B_Buffer: 0.1 M PiNa, pH 7.0, flow rate 0.6 ml/min, gradient A50% B50%→A0 %
B100%, 12 min (data collection 15 min), column temperature at room temperature, thermostat temperature at room temperature, detector at wavelength of 280 nm.

The samples were reacted under the following conditions.
a: Trastuzumab raw material b: Trastuzumab + peptide disulfide linker conjugate-NHS activated form 30 equivalents;

Retension Time 6.8590 min is considered to be the trastuzumab raw material and 8.0655 min is the peptide derived peak (Figure 42).

[Example 32: Synthesis of compound (peptide disulfide linker conjugate-NHS activator) having affinity substance for soluble protein, cleavable moiety and reactive group, and modification of anti-HER2 IgG antibody trastuzumab using the compound and its analysis]
(32-1) Synthesis of IgG1 Fc-Binding Peptide The sequence of Ac-RGNCAYHLGQLVWCTKH-NH ₂ (SEQ ID NO: 58) was synthesized in the same manner as in Example 1 by the Fmoc solid-phase synthesis method. The desired product (36.7 mg, 18.1 μmol) was obtained.

MS (ESI) m/z: z=3 676.75 [M+3H] ^<3+> , z=4 507.80 [M+4H] ^<4+>

上記アミノ酸配列は、配列番号５８である。 (32-2) Formation of intramolecular disulfide bond at Cys at positions 4 and 14 of Ac-RGNCAYHLGQLVWCTKH-NH ₂ (SEQ ID NO: 58)

The amino acid sequence is SEQ ID NO:58.

The peptide (36.7 mg, 18.1 μmol) synthesized in (32-1) was dissolved in DMSO (5.00 mL), 2M NH ₃ -MeOH (18.1 μL, 36.2 μmol), hydrogen peroxide solution (37 .0 μL, 362 μmol) was added and stirred at room temperature for 20 hours. A 2M trifluoroacetic acid aqueous solution was added to the reaction solution to stop the reaction, and the solution was dissolved in a 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. , eluted with a mixed solution of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain the above peptide (18.2 mg, 8.99 μmol).

MS (ESI) m/z: z=3 676.00 [M+3H] ^<3+> , z=4 507.30 [M+4H] ^<4+>

上記アミノ酸配列は、配列番号５８である。 (32-3) Linking disulfide linker and peptide

The amino acid sequence is SEQ ID NO:58.

Ac-RGNCAYHLGQLVWCTKH-NH ₂ (SEQ ID NO: 58) synthesized in (32-2) (18.2 mg, 8.99 μmol, provided that two cysteines at positions 4 and 14 each form an intramolecular disulfide bond. ) was dissolved in N,N-dimethylformamide (1.00 mL), and di(N-succinimidyl) 3,3′-dithiodipropionate (145 mg, 360 μmol) was dissolved in acetonitrile (1.50 mL). The mixture was added and stirred at room temperature for 24 hours. After removing acetonitrile by concentrating under reduced pressure, this was dissolved in 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. Elution was performed with a mixed solution of water and acetonitrile containing 0.05% acetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain the peptide disulfide linker conjugate-NHS activated product (3.00 mg, 1.30 μmol). .

MS (ESI) m/z: z=3 772.45 [M+3H] ³⁺
HPLC Purity: 90%

(32-4) Specific modification of anti-HER2 IgG antibody trastuzumab and analysis by ESI-TOFMS The peptide disulfide linker conjugate-NHS activator synthesized in (32-3) is N,
It was dissolved in N'-dimethylformamide to 21.6 mM. Dissolve 500 μg of anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical Co., Ltd.) in 46.9 μL of 60 mM sodium acetate buffer (pH 4.7), add 4.7 μL of 21.6 mM peptide reagent (30 equivalents to the antibody), and warm to room temperature. and stirred for 1 hour. The reaction solution was added to a NAP-5 column to stop the reaction and replaced with 20 mM PBS buffer. When the mass was measured by ESI-TOFMS, a peak was observed at 148077 for the raw material trastuzumab and a peak at 150281 for the product into which one binding peptide was introduced.

(32-5) HIC-UPLC Analysis of Specific Modification of Anti-HER2 IgG Antibody Trastuzumab The antibody/peptide complex produced in (32-4) and the starting antibody were analyzed by HIC. A Protein-Pak Hi Res HIC Column (Waters) 4.6×100 mm 2.5 μm was used as the column. A_Buffer: 0.1 M PiNa, 2.3 M (NH ₄ ) ₂ SO ₄ , pH 7.0, B_Buffer: 0.1 M PiNa, pH 7.0, flow rate 0.6 ml/min, gradient A60% B40%→A0 %
B100%, 16 min (20 min of data collection), column temperature of 40° C., thermostat temperature of 40° C., detector at wavelength of 280 nm.

The samples were reacted under the following conditions.
a: Trastuzumab raw material b: Trastuzumab + peptide disulfide linker conjugate-NHS activated form 30 equivalents;

Retension Time 11.1800 minutes is considered to be the trastuzumab raw material, and 11.8682 minutes is considered to be a compound in which one peptide was introduced into trastuzumab (Fig. 43).

[Example 33: Synthesis of a compound (peptide disulfide linker conjugate-NHS activator) having an affinity substance for soluble proteins, a cleavable moiety and a reactive group, and modification of the anti-HER2 IgG antibody trastuzumab using the compound and its analysis]
(33-1) Synthesis of IgG1 Fc-Binding Peptide The sequence of Ac-RGNCAYHLGQLVWCTYK-NH ₂ (SEQ ID NO: 59) was synthesized in the same manner as in Example 1 by the Fmoc solid-phase synthesis method. The desired product (52.1 mg, 25.4 μmol) was obtained.

MS (ESI) m/z: z=2 1027.3 [M+2H] ^<2+> , z=3 685.45 [M+3H] ^<3+>

上記アミノ酸配列は、配列番号５９である。 (33-2) Formation of intramolecular disulfide bond at Cys at positions 4 and 14 of Ac-RGNCAYHLGQLVWCTYK-NH ₂ (SEQ ID NO: 59)

The amino acid sequence is SEQ ID NO:59.

The peptide (52.1 mg, 25.4 µmol) synthesized in (33-1) was dissolved in DMSO (5.00 mL), and 2M NH ₃ -MeOH (25.4 µL, 50.8 µmol),
A hydrogen peroxide solution (51.9 μL, 508 μmol) was added, and the mixture was stirred at room temperature for 20 hours. A 2M trifluoroacetic acid aqueous solution was added to the reaction solution to stop the reaction, and the solution was dissolved in a 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. , eluted with a mixed solution of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain the above peptide (4.70 mg, 2.29 μmol).

MS (ESI) m/z: z=2 1026.5 [M+2H] ^<2+> , z=3 684.75 [M+3H] ^<3+>

上記アミノ酸配列は、配列番号５９である。 (33-3) Linking disulfide linker and peptide

The amino acid sequence is SEQ ID NO:59.

Ac-RGNCAYHLGQLVWCTYK-NH ₂ (SEQ ID NO: 59) synthesized in (33-2) (4.70 mg, 2.29 μmol, provided that two cysteines at positions 4 and 14 each form an intramolecular disulfide bond. ) was dissolved in N,N-dimethylformamide (0.50 mL), and di(N-succinimidyl) 3,3′-dithiodipropionate (37.0 mg, 91.6 μmol) was added to acetonitrile (1.00 mL). The dissolved solution was added and stirred at room temperature for 24 hours. After removing acetonitrile by concentrating under reduced pressure, this was dissolved in 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. Elution was performed with a mixed solution of water and acetonitrile containing 0.05% acetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain the peptide disulfide linker conjugate-NHS activated product (5.00 mg, 2.14 μmol). .

MS (ESI) m/z: z=3 781.20 [M+3H] ³⁺
HPLC Purity: 79%

(33-4) Specific modification of anti-HER2 IgG antibody trastuzumab and analysis by ESI-TOFMS The peptide disulfide linker conjugate-NHS-activated product synthesized in (33-3) was dissolved in N,N'-dimethylformamide. 6 mM. Dissolve 500 μg of anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical Co., Ltd.) in 46.9 μL of 60 mM sodium acetate buffer (pH 4.7), add 4.7 μL of 21.6 mM peptide reagent (30
equivalent volume) and stirred at room temperature for 1 hour. The reaction solution was added to a NAP-5 column to stop the reaction and replaced with 20 mM PBS buffer. When the mass was measured by ESI-TOFMS, a peak was observed at 148223 for the starting trastuzumab and a peak at 150488 for the product into which one binding peptide was introduced.

(33-5) HIC-UPLC Analysis of Specific Modification of Anti-HER2 IgG Antibody Trastuzumab The antibody/peptide complex produced in (33-4) and the starting antibody were analyzed by HIC. A Protein-Pak Hi Res HIC Column (Waters) 4.6×100 mm 2.5 μm was used as the column. A_Buffer: 0.1 M PiNa, 2.3 M (NH ₄ ) ₂ SO ₄ , pH 7.0, B_Buffer: 0.1 M PiNa, pH 7.0, flow rate 0.6 ml/min, gradient A60% B40%→A0 %
B100%, 16 min (20 min of data collection), column temperature of 40° C., thermostat temperature of 40° C., detector at wavelength of 280 nm.

The samples were reacted under the following conditions.
a: Trastuzumab raw material b: Trastuzumab + peptide disulfide linker conjugate-NHS activated form 30 equivalents;

Retension Time 11.1867 minutes is considered to be the trastuzumab raw material, and 12.2550 minutes is considered to be a compound in which one peptide was introduced into trastuzumab (Fig. 44).

[Example 34 (peptide disulfide linker conjugate-NHS activator) synthesis, modification of anti-HER2 IgG antibody trastuzumab using the compound, and analysis thereof]
(34-1) Synthesis of IgG1 Fc-Binding Peptide The sequence of Ac-RGNCAYHRGQLVWCTKH-NH ₂ (SEQ ID NO: 60) was synthesized in the same manner as in Example 1 by the Fmoc solid-phase synthesis method. The desired product (66.3 mg, 32.0 μmol) was obtained.

MS (ESI) m/z: z=3 691.10 [M+3H] ^<3+> , z=4 518.55 [M+4H] ^<4+>

上記アミノ酸配列は、配列番号６０である。 (34-2) Formation of intramolecular disulfide bond at Cys at positions 4 and 14 of Ac-RGNCAYHRGQLVWCTKH-NH ₂ (SEQ ID NO: 60)

The amino acid sequence is SEQ ID NO:60.

The peptide (66.3 mg, 32.0 μmol) synthesized in (34-1) was dissolved in DMSO (5.00 mL), 2M NH ₃ -MeOH (32.0 μL, 64.0 μmol), hydrogen peroxide solution (65 .4 μL, 640 μmol) was added and stirred at room temperature for 20 hours. A 2M trifluoroacetic acid aqueous solution was added to the reaction solution to stop the reaction, and the solution was dissolved in a 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. , eluted with a mixed solution of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain the above peptide (40.0 mg, 19.3 μmol).

MS (ESI) m/z: z=3 690.40 [M+3H] ^<3+> , z=4 518.05 [M+4H] ^<4+>

上記アミノ酸配列は、配列番号６０である。 (34-3) Linking disulfide linker and peptide

The amino acid sequence is SEQ ID NO:60.

Ac-RGNCAYHRGQLVWCTKH-NH ₂ (SEQ ID NO: 60) synthesized in (34-2) (40.0 mg, 19.3 μmol, provided that the two cysteines at positions 4 and 14 each form an intramolecular disulfide bond. ) was dissolved in N,N-dimethylformamide (1.00 mL), and di(N-succinimidyl) 3,3′-dithiodipropionate (312 mg, 772 μmol) was dissolved in acetonitrile (2.00 mL). The mixture was added and stirred at room temperature for 24 hours. After removing acetonitrile by concentrating under reduced pressure, this was dissolved in a 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. Elution was performed with a mixed solution of water and acetonitrile containing 0.05% acetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain the peptide disulfide linker conjugate-NHS activated form (4.50 mg, 1.91 μmol). .

MS (ESI) m/z: z=3 786.85 [M+3H] ^<3+> , m/z: z=4 590.45 [M+4H] ^<4+>
HPLC Purity: 78%

(34-4) Specific modification of anti-HER2 IgG antibody trastuzumab and analysis by ESI-TOFMS The peptide disulfide linker conjugate-NHS-activated product synthesized in (34-3) was dissolved in N,N'-dimethylformamide. 6 mM. Dissolve 500 μg of anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical Co., Ltd.) in 46.9 μL of 60 mM sodium acetate buffer (pH 4.7), add 4.7 μL of 21.6 mM peptide reagent (30 equivalents to the antibody), and warm to room temperature. and stirred for 1 hour. The reaction solution was added to a NAP-5 column to stop the reaction and replaced with 20 mM PBS buffer. When the mass was measured by ESI-TOFMS, a peak was observed at 148081 for the raw material trastuzumab.

(34-5) Confirmation of heavy chain selectivity by ESI-TOFMS analysis under reducing conditions of a specific modification of trastuzumab 7 mM tris(2-carboxyethyl)phosphine on the antibody/peptide complex produced in (34-4) 5 μL of hydrochloride solution (equal volume to antibody) was added and stirred at room temperature for 20 minutes. When the mass was measured by ESI-TOFMS, the light chain peaks were observed at 50682 and 50844 where a thiopropionyl group was introduced into the heavy chain and at 23439 which was the same as the starting material.

(34-6) HIC-UPLC Analysis of Specific Modification of Anti-HER2 IgG Antibody Trastuzumab The antibody/peptide complex produced in (34-4) and the starting antibody were analyzed by HIC. A Protein-Pak Hi Res HIC Column (Waters) 4.6×100 mm 2.5 μm was used as the column. A_Buffer: 0.1 M PiNa, 2.3 M (NH ₄ ) ₂ SO ₄ , pH 7.0, B_Buffer: 0.1 M PiNa, pH 7.0, flow rate 0.6 ml/min, gradient A60% B40%→A0 %
B100%, 16 min (20 min of data collection), column temperature of 40° C., thermostat temperature of 40° C., detector at wavelength of 280 nm.

The samples were reacted under the following conditions.
a: Trastuzumab raw material b: Trastuzumab + peptide disulfide linker conjugate-NHS activated form 30 equivalents;

The Retension Time 10.0610 minutes is believed to be a compound derived from the trastuzumab source. A peak was also confirmed at 11.0666 minutes (Fig. 45).

[Example 35: Synthesis of a compound (peptide disulfide linker conjugate-NHS activator) having an affinity substance for soluble proteins, a cleavable moiety and a reactive group, and modification of the anti-HER2 IgG antibody trastuzumab using the compound and its analysis]
(35-1) Synthesis of IgG1 Fc-Binding Peptide The sequence Ac-KNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC-NH ₂ (SEQ ID NO: 61) was synthesized by Fmoc solid-phase synthesis. Liberty Blue manufactured by CEM was used as a peptide synthesizer. All reagents used are manufactured by Watanabe Chemical Industry. Resin was Fmoc-NH-SAL-PEG Resin HL, arginine (R), cysteine (C) and histidine (H) were double-coupled. Cutting from Resin was performed in a solution of trifluoroacetic acid:water:triisopropylsilane:ethanedithiol=94:2.5:1.0:2.5 under stirring conditions for 3 hours. After cutting out, the resin was removed by filtration, and the trifluoroacetic acid was removed. Diethyl ether was added to the resulting crystals for ether precipitation, and the resulting white crystals were collected by filtration. This was dissolved in an aqueous 0.05% trifluoroacetic acid solution and subjected to reversed-phase high performance liquid chromatography using octadodecyl chemically bonded silica gel as a packing material. Elution was performed with the mixed solution, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain the desired product (47.0 mg, 11.0 μmol).

MS (ESI) m/z: z=5 853.35 [M+5H] ^<5+> , z=6 711.25 [M+6H] ^<6+>

上記アミノ酸配列は、配列番号６１である。 (35-2) Formation of intramolecular disulfide bond at Cys at positions 5 and 34 of Ac-KNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC-NH ₂ (SEQ ID NO: 61)

The amino acid sequence is SEQ ID NO:61.

The peptide (47.0 mg, 11.0 μmol) synthesized in (35-1) was dissolved in DMSO (1.10 mL), and 0.1 M Tris-HCl pH 8.0 (11.0 mL) was added. Glutathione oxidized form (34.0 mg, 55.0 μmol) was added to this solution and stirred at room temperature for 20 hours. A 2M trifluoroacetic acid aqueous solution was added to the reaction solution to stop the reaction, and the solution was dissolved in a 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. , eluted with a mixed solution of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain the desired product (18.1 mg, 4.25 μmol).

MS (ESI) m/z: z=5 852.95 [M+5H] ^<5+> , z=6 710.95 [M+6H] ^<6+>

上記アミノ酸配列は、配列番号６１である。 (35-3) Linking disulfide linker and peptide

The amino acid sequence is SEQ ID NO:61.

Ac-KNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC-NH ₂ (SEQ ID NO: 61) synthesized in (35-2) (18.1 mg, 4.25 μmol, provided that two cysteines at positions 5 and 34 each form an intramolecular disulfide bond ) was dissolved in N,N-dimethylformamide (1.00 mL), and di(N-succinimidyl) 3,3′-dithiodipropionate (69.0 mg, 0.170 mmol) was added to acetonitrile (2.00 mL). The dissolved solution was added and stirred at room temperature for 24 hours. After removing acetonitrile by concentrating under reduced pressure, this was dissolved in 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. Elution was performed with a mixed solution of water and acetonitrile containing 0.05% acetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain the peptide disulfide linker conjugate-NHS activated product (4.00 mg, 0.80 μmol). .

MS (ESI) m/z: z=5 910.85 [M+5H] ^<5+> , z=6 759.20 [M+6H] ^<6+>
HPLC Purity: 60%

(35-4) Specific modification of anti-HER2 IgG antibody trastuzumab and analysis by ESI-TOFMS The peptide disulfide linker conjugate-NHS-activated product synthesized in (35-3) was dissolved in N,N'-dimethylformamide and bottom. Dissolve 500 µg of anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical Co., Ltd.) in 171 µL of 50 mM sodium acetate buffer (pH 5.5), add 8.5 µL of 4 mM peptide reagent (10 equivalents to the antibody), and stir at room temperature for 1 hour. bottom. The reaction solution was added to a NAP-5 column to stop the reaction and replaced with 20 mM PBS buffer. When the mass was measured by ESI-TOFMS, a peak was observed at 148234 for the raw material trastuzumab, a peak was observed at 152672 for the product into which one binding peptide was introduced, and two binding peptides were introduced. A peak was confirmed at 157,107.

(35-5) Confirmation of heavy chain selectivity by ESI-TOFMS analysis under reducing conditions of a specific modification of trastuzumab 7 mM tris(2-carboxyethyl)phosphine on the antibody/peptide complex produced in (35-4) 5 μL of hydrochloride solution (equal volume to antibody) was added and stirred at room temperature for 20 minutes. When the mass was measured by ESI-TOFMS, peaks were observed at 50684 and 50844 where a thiopropionyl group was introduced into the heavy chain of the product.

(35-5) HIC-UPLC Analysis of Specific Modification of Anti-HER2 IgG Antibody Trastuzumab The antibody/peptide complex produced in (35-4) and the starting antibody were analyzed by HIC. A Protein-Pak Hi Res HIC Column (Waters) 4.6×100 mm 2.5 μm was used as the column. A_Buffer: 0.1 M PiNa, 2.3 M (NH ₄ ) ₂ SO ₄ , pH 7.0, B_Buffer: 0.1 M PiNa, pH 7.0, flow rate 0.6 ml/min, gradient A60% B40%→A0 %
B100%, 16 min (20 min of data collection), column temperature of 40° C., thermostat temperature of 40° C., detector at wavelength of 280 nm.

The samples were reacted under the following conditions.
a: Trastuzumab raw material b: Trastuzumab + peptide disulfide linker conjugate-NHS activated form 10 equivalents;

Retension Time 10.0542 minutes is considered to be the trastuzumab raw material, 9.4481 minutes to the compound into which one peptide was introduced, and 8.5522 minutes to the compound into which two peptides were introduced (Fig. 46).

[Example 36: Synthesis of a compound (peptide disulfide linker conjugate-NHS activator) having an affinity substance for soluble proteins, a cleavable moiety and a reactive group, and modification of the anti-HER2 IgG antibody trastuzumab using the compound and its analysis]
(36-1) Synthesis of IgG1 Fc-Binding Peptide The sequence of Ac-FNMQCQKRFYEALHDPNLNEEQRNARIRSIRDDC-NH ₂ (SEQ ID NO: 62) was synthesized by the Fmoc solid-phase synthesis method. Liberty Blue manufactured by CEM was used as a peptide synthesizer. All reagents used are manufactured by Watanabe Chemical Industry. Resin was Fmoc-NH-SAL-PEG Resin HL, arginine (R), cysteine (C) and histidine (H) were double-coupled. Cutting from Resin was performed in a solution of trifluoroacetic acid:water:triisopropylsilane:ethanedithiol=94:2.5:1.0:2.5 under stirring conditions for 3 hours. After cutting out, the resin was removed by filtration, and the trifluoroacetic acid was removed. Diethyl ether was added to the resulting crystals for ether precipitation, and the resulting white crystals were collected by filtration. This was dissolved in an aqueous 0.05% trifluoroacetic acid solution and subjected to reversed-phase high performance liquid chromatography using octadodecyl chemically bonded silica gel as a packing material. Elution was performed with the mixed solution, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain the desired product (76.3 mg, 18.0 μmol).

MS (ESI) m/z: z=5 851.45 [M+5H] ^<5+> , z=6 709.75 [M+6H] ^<6+>

上記アミノ酸配列は、配列番号６２である。 (36-2) Formation of intramolecular disulfide bond at Cys at positions 5 and 34 of Ac-FNMQCQKRFYEALHDPNLNEEQRNARIRSIRDDC-NH ₂ (SEQ ID NO: 62)

The amino acid sequence is SEQ ID NO:62.

The peptide (76.3 mg, 18.0 μmol) synthesized in (36-1) was dissolved in DMSO (1.80 mL), and 0.1 M Tris-HCl pH 8.0 (18.0 mL) was added. Glutathione oxidized form (55.0 mg, 89.5 μmol) was added to this solution and stirred at room temperature for 20 hours. A 2M trifluoroacetic acid aqueous solution was added to the reaction solution to stop the reaction, and the solution was dissolved in a 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. , trifluoroacetic acid 0.05%
and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain the desired product (28.5 mg, 6.71 μmol).

MS (ESI) m/z: z=5 851.05 [M+5H] ^<5+> , z=6 709.40 [M+6H] ^<6+>

上記アミノ酸配列は、配列番号６２である。 (36-3) Linking disulfide linker and peptide

The amino acid sequence is SEQ ID NO:62.

Ac-FNMQCQKRFYEALHDPNLNEEQRNARIRSIRDDC-NH ₂ (SEQ ID NO: 62) synthesized in (36-2) (28.5 mg, 6.71 μmol, provided that two cysteines at positions 5 and 34 each form an intramolecular disulfide bond) ) was dissolved in N,N-dimethylformamide (1.00 mL), and di(N-succinimidyl) 3,3′-dithiodipropionate (108 mg, 0.268 mmol) was dissolved in acetonitrile (2.00 mL). was added and stirred at room temperature for 24 hours. After removing acetonitrile by concentrating under reduced pressure, this was dissolved in 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. Elution was performed with a mixed solution of water and acetonitrile containing 0.05% acetic acid, and each fraction was confirmed by LC-MS. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain the peptide disulfide linker conjugate-NHS activated product (7.10 mg, 1.56 μmol). .

MS (ESI) m/z: z=5 909.05 [M+5H] ^<5+> , z=6 757.70 [M+6H] ^<6+>
HPLC Purity: 87%

(36-4) Specific modification of anti-HER2 IgG antibody trastuzumab and analysis by ESI-TOFMS The peptide disulfide linker conjugate-NHS-activated product synthesized in (36-3) was dissolved in N,N'-dimethylformamide. 6 mM. Dissolve 500 μg of anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical Co., Ltd.) in 46.9 μL of 10 mM sodium acetate buffer (pH 5.5), add 4.7 μL of 21.6 mM peptide reagent (30 equivalents to the antibody), and warm to room temperature. and stirred for 1 hour. The reaction solution was added to a NAP-5 column to stop the reaction and replaced with 20 mM PBS buffer. When the mass was measured by ESI-TOFMS, a peak was observed at 148232 for the starting trastuzumab and a peak at 152664 for the product into which one binding peptide was introduced.

(36-5) Confirmation of heavy chain selectivity by ESI-TOFMS analysis under reducing conditions of a specific modification of trastuzumab 7 mM tris(2-carboxyethyl)phosphine on the antibody/peptide complex produced in (36-4) 5 μL of hydrochloride solution (equal volume to antibody) was added and stirred at room temperature for 20 minutes. When the mass was measured by ESI-TOFMS, a peak was observed at 50681 where a thiopropionyl group was introduced into the heavy chain of the product.

(36-6) HIC-UPLC Analysis of Specific Modification of Anti-HER2 IgG Antibody Trastuzumab The antibody/peptide complex produced in (36-4) and the starting antibody were analyzed by HIC. A Protein-Pak Hi Res HIC Column (Waters) 4.6×100 mm 2.5 μm was used as the column. A_Buffer: 0.1 M PiNa, 2.3 M (NH ₄ ) ₂ SO ₄ , pH 7.0, B_Buffer: 0.1 M PiNa, pH 7.0, flow rate 0.6 ml/min, gradient A60% B40%→A0 %
B100%, 16 min (20 min of data collection), column temperature of 40° C., thermostat temperature of 40° C., detector at wavelength of 280 nm.

The samples were reacted under the following conditions.
a: Trastuzumab raw material b: Trastuzumab + peptide disulfide linker conjugate-NHS activated form 30 equivalents;

Retension Time 9.6620 minutes is considered to be the trastuzumab raw material, and 10.3237 minutes is considered to be a compound into which one peptide has been introduced (Fig. 47).

[Example 37: Synthesis of a compound (peptide disulfide linker conjugate-NHS activator) having an affinity substance for soluble proteins, a cleavable moiety and a reactive group, and modification of the anti-HER2 IgG antibody trastuzumab using the compound and its analysis]
(37-1) Synthesis of IgG1 Fc-Binding Peptide The sequence Ac-FNMQCQRRFYEAKHDPNLNEEQRNARIRSIRDDC-NH ₂ (SEQ ID NO: 63) was synthesized by Fmoc solid-phase synthesis. Liberty Blue manufactured by CEM was used as a peptide synthesizer. All reagents used are manufactured by Watanabe Chemical Industry. Resin was Fmoc-NH-SAL-PEG Resin HL, arginine (R), cysteine (C) and histidine (H) were double-coupled. Cutting from Resin was performed in a solution of trifluoroacetic acid:water:triisopropylsilane:ethanedithiol=94:2.5:1.0:2.5 under stirring conditions for 3 hours. After cutting out, the resin was removed by filtration, and the trifluoroacetic acid was removed. Diethyl ether was added to the resulting crystals for ether precipitation, and the resulting white crystals were collected by filtration. This was dissolved in an aqueous 0.05% trifluoroacetic acid solution and subjected to reversed-phase high performance liquid chromatography using octadodecyl chemically bonded silica gel as a packing material. Elute with the mixed solution and separate each fraction by LC-M.
Confirmed by S. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain the desired product (67.4 mg, 15.7 μmol).

MS (ESI) m/z: z=5 860.10 [M+5H] ^<5+> , z=6 716.95 [M+6H] ^<6+>

上記アミノ酸配列は、配列番号６３である。 (37-2) Formation of an intramolecular disulfide bond at Cys at positions 5 and 34 of Ac-FNMQCQRRFYEAKHDPNLNEEQRNARIRSIRDDC-NH ₂ (SEQ ID NO: 63)

The amino acid sequence is SEQ ID NO:63.

The peptide (67.4 mg, 15.7 μmol) synthesized in (37-1) was dissolved in DMSO (1.50 mL), and 0.1 M Tris-HCl pH 8.0 (15.0 mL) was added. Glutathione oxidized form (48.0 mg, 78.5 μmol) was added to this solution and stirred at room temperature for 20 hours. A 2M trifluoroacetic acid aqueous solution was added to the reaction solution to stop the reaction, and the solution was dissolved in a 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. , eluted with a mixed solution of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain the desired product (16.1 mg, 3.75 μmol).

MS (ESI) m/z: z=5 859.75 [M+5H] ^<5+> , z=6 716.60 [M+6H] ^<6+>

上記アミノ酸配列は、配列番号６３である。 (37-3) Linking disulfide linker and peptide

The amino acid sequence is SEQ ID NO:63.

Ac-FNMQCQRRFYEAKHDPNLNEEQRNARIRSIRDDC-NH ₂ (SEQ ID NO: 63) synthesized in (37-2) (16.1 mg, 3.75 μmol, provided that two cysteines at positions 5 and 34 each form an intramolecular disulfide bond) ) was dissolved in N,N-dimethylformamide (1.00 mL), and di(N-succinimidyl) 3,3′-dithiodipropionate (61.0 mg, 0.150 mmol) was dissolved in acetonitrile (2.00 mL). The resulting solution was added and stirred at room temperature for 24 hours. After removing acetonitrile by concentrating under reduced pressure, this was dissolved in 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. Elution was performed with a mixed solution of water and acetonitrile containing 0.05% acetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain the peptide disulfide linker conjugate-NHS activated product (5.30 mg, 1.16 μmol). .

MS (ESI) m/z: z=5 917.60 [M+5H] ^<5+> , z=6 764.85 [M+6H] ^<6+>
HPLC Purity: 54%

(37-4) Specific modification of anti-HER2 IgG antibody trastuzumab and analysis by ESI-TOFMS The peptide disulfide linker conjugate-NHS-activated product synthesized in (34-3) was dissolved in N,N'-dimethylformamide. 6 mM. Dissolve 500 μg of anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical Co., Ltd.) in 46.9 μL of 10 mM sodium acetate buffer (pH 5.5), add 4.7 μL of 21.6 mM peptide reagent (30 equivalents to the antibody), and warm to room temperature. and stirred for 1 hour. The reaction solution was added to a NAP-5 column to stop the reaction and replaced with 20 mM PBS buffer. When the mass was measured by ESI-TOFMS, a peak was observed at 148238 for the raw material trastuzumab and a peak at 152703 for the product into which one binding peptide was introduced.

(37-5) Confirmation of heavy chain selectivity by ESI-TOFMS analysis under reducing conditions of a specific modification of trastuzumab 7 mM tris(2-carboxyethyl)phosphine on the antibody/peptide complex produced in (37-4) 5 μL of hydrochloride solution (equal volume to antibody) was added and stirred at room temperature for 20 minutes. When the mass was measured by ESI-TOFMS, a peak was observed at 50682 where a thiopropionyl group was introduced into the heavy chain of the product.

(37-6) HIC-UPLC Analysis of Specific Modification of Anti-HER2 IgG Antibody Trastuzumab The antibody/peptide complex produced in (37-4) and the starting antibody were analyzed by HIC. A Protein-Pak Hi Res HIC Column (Waters) 4.6×100 mm 2.5 μm was used as the column. A_Buffer: 0.1 M PiNa, 2.3 M (NH ₄ ) ₂ SO ₄ , pH 7.0, B_Buffer: 0.1 M PiNa, pH 7.0, flow rate 0.6 ml/min, gradient A60% B40%→A0 %
B100%, 16 min (20 min of data collection), column temperature of 40° C., thermostat temperature of 40° C., detector at wavelength of 280 nm.

The samples were reacted under the following conditions.
a: Trastuzumab raw material b: Trastuzumab + peptide disulfide linker conjugate-NHS activated form 30 equivalents;

Retension Time 9.5802 minutes is considered to be the trastuzumab raw material, and 9.1031 minutes is considered to be a compound into which one peptide has been introduced (Fig. 48).

[Example 38: Synthesis of a compound (peptide disulfide linker conjugate-NHS activator) having an affinity substance for soluble proteins, a cleavable moiety and a reactive group, and modification of the anti-HER2 IgG antibody trastuzumab using the compound and its analysis]
(38-1) Synthesis of IgG1 Fc-Binding Peptide The sequence of Ac-FNMQCQRRFYEALHDPNLNEEQRKARIRSIRDDC-NH ₂ (SEQ ID NO: 64) was synthesized in the same manner as in Example 1 by the Fmoc solid-phase synthesis method. got the target.

上記アミノ酸配列は、配列番号６４である。 (38-2) Formation of intramolecular disulfide bond at Cys at positions 5 and 34 of Ac-FNMQCQRRFYEALHDPNLNEEQRKARIRSIRDDC-NH ₂ (SEQ ID NO: 64)

The amino acid sequence is SEQ ID NO:64.

The peptide synthesized in (38-1) was dissolved in DMSO, and 0.1M Tris-HCl pH 8.0 was added. Glutathione oxidized form was added to this solution and stirred at room temperature for 20 hours. A 2M trifluoroacetic acid aqueous solution was added to the reaction solution to stop the reaction, and the solution was dissolved in a 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. , eluted with a mixed solution of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain the desired product (20.0 mg, 4.66 μmol).

MS (ESI) m/z: z = 4 1074.15 [M+4H] ⁴⁺ , z = 5 859.65 [M+5H] ⁵⁺

上記アミノ酸配列は、配列番号６４である。 (38-3) Linking disulfide linker and peptide

The amino acid sequence is SEQ ID NO:64.

Ac-FNMQCQRRFYEALHDPNLNEEQRKARIRSIRDDC-NH ₂ (SEQ ID NO: 64) synthesized in (38-2) (20.0 mg, 4.66 μmol, provided that two cysteines at positions 5 and 34 each form an intramolecular disulfide bond ) was dissolved in N,N-dimethylformamide (1.00 mL), and di(N-succinimidyl) 3,3′-dithiodipropionate (75.4 mg, 186 μmol) was dissolved in acetonitrile (1.00 mL). was added and stirred at room temperature for 24 hours. After removing acetonitrile by concentrating under reduced pressure, this was dissolved in 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. Elution was performed with a mixed solution of water and acetonitrile containing 0.05% acetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain the peptide disulfide linker conjugate-NHS activated form (9.00 mg, 1.96 μmol). .

MS (ESI) m/z: z=5 917.45 [M+5H] ^<5+> , z=6 764.65 [M+6H] ^<6+>
HPLC Purity: 100%

(38-4) Specific modification of anti-HER2 IgG antibody trastuzumab and analysis by ESI-TOFMS The peptide disulfide linker conjugate-NHS-activated product synthesized in (38-3) was dissolved in N,N'-dimethylformamide and bottom. Dissolve 500 µg of anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical Co., Ltd.) in 171 µL of 50 mM sodium acetate buffer (pH 5.5), add 8.5 µL of 4 mM peptide reagent (10 equivalents to the antibody), and stir at room temperature for 1 hour. bottom. The reaction solution was added to a NAP-5 column to stop the reaction and replaced with 20 mM PBS buffer. When the mass was measured by ESI-TOFMS, a peak was observed at 148070 for the raw material trastuzumab, a peak was observed at 152538 for the product into which one binding peptide was introduced, and two binding peptides were introduced. A peak was observed at 157,165.

(38-5) Confirmation of heavy chain selectivity by ESI-TOFMS analysis under reducing conditions of a specific modification of trastuzumab 7 mM tris(2-carboxyethyl)phosphine on the antibody/peptide complex produced in (38-4) 5 μL of hydrochloride solution (equal volume to antibody) was added and stirred at room temperature for 20 minutes. When the mass was measured by ESI-TOFMS, light chain peaks were observed at 50685 and 50847 where thiopropionyl was introduced into the heavy chain and at 23439 which was the same as the starting material.

(38-6) HIC-UPLC Analysis of Specific Modification of Anti-HER2 IgG Antibody Trastuzumab The antibody/peptide complex produced in (38-4) and the starting antibody were analyzed by HIC. A Protein-Pak Hi Res HIC Column (Waters) 4.6×100 mm 2.5 μm was used as the column. A_Buffer: 0.1 M PiNa, 2.3 M (NH ₄ ) ₂ SO ₄ , pH 7.0, B_Buffer: 0.1 M PiNa, pH 7.0, flow rate 0.6 ml/min, gradient A60% B40%→A0 %
B100%, 16 min (20 min of data collection), column temperature of 40° C., thermostat temperature of 40° C., detector at wavelength of 280 nm.

The samples were reacted under the following conditions.
a: Trastuzumab raw material b: Trastuzumab + peptide disulfide linker conjugate-NHS activated form 10 equivalents;

Retension Time 9.7820 minutes is considered to be the trastuzumab raw material, 9.3370 minutes to the compound into which one peptide was introduced, and 8.7313 minutes to the compound into which two peptides were introduced (Fig. 49).

[Example 39: Synthesis of a compound (peptide disulfide linker conjugate-NHS activator) having an affinity substance for soluble proteins, a cleavable moiety and a reactive group, and modification of the anti-HER2 IgG antibody trastuzumab using the compound and its analysis]
(39-1) Synthesis of IgG1 Fc-Binding Peptide The sequence of Ac-FNMQCQRRFYEALHDPNLNKEQRNARIRSIRDDC-NH ₂ (SEQ ID NO: 65) was synthesized in the same manner as in Example 1 by the Fmoc solid-phase synthesis method. got the target.

上記アミノ酸配列は、配列番号６５である。 (39-2) Formation of an intramolecular disulfide bond at Cys at positions 5 and 34 of Ac-FNMQCQRRFYEALHDPNLNKEQRNARIRSIRDDC-NH ₂ (SEQ ID NO: 65)

The amino acid sequence is SEQ ID NO:65.

The peptide synthesized in (39-1) was dissolved in DMSO, and 0.1M Tris-HCl pH 8.0 was added. Glutathione oxidized form was added to this solution and stirred at room temperature for 20 hours. A 2M trifluoroacetic acid aqueous solution was added to the reaction solution to stop the reaction, and the solution was dissolved in a 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. , eluted with a mixed solution of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain the desired product (23.0 mg, 5.38 μmol).

MS (ESI) m/z: z = 4 1070.55 [M+4H] ⁴⁺ , z = 5 856.55 [M+5H] ⁵⁺

上記アミノ酸配列は、配列番号６５である。 (39-3) Linking disulfide linker and peptide

The amino acid sequence is SEQ ID NO:65.

Ac-FNMQCQRRFYEALHDPNLNKEQRNARIRSIRDDC-NH ₂ (SEQ ID NO: 65) synthesized in (39-2) (23.0 mg, 5.38 μmol, provided that two cysteines at positions 5 and 34 each form an intramolecular disulfide bond) ) was dissolved in N,N-dimethylformamide (1.00 mL), and di(N-succinimidyl) 3,3′-dithiodipropionate (87.0 mg, 215 μmol) was dissolved in acetonitrile (1.00 mL). was added and stirred at room temperature for 24 hours. After removing acetonitrile by concentrating under reduced pressure, this was dissolved in 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. Elution was performed with a mixed solution of water and acetonitrile containing 0.05% acetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain the peptide disulfide linker conjugate-NHS activated product (16.0 mg, 3.50 μmol). .

MS (ESI) m/z: z = 5 914.45 [M+5H] ⁵⁺ , z = 6 762.15 [M+6H] ⁶⁺
HPLC Purity: 83%

(39-4) Specific modification of anti-HER2 IgG antibody trastuzumab and analysis by ESI-TOFMS The peptide disulfide linker conjugate-NHS-activated product synthesized in (39-3) was dissolved in N,N'-dimethylformamide and bottom. Dissolve 500 μg of anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical Co., Ltd.) in 171 μL of 50 mM sodium acetate buffer (pH 5.5), add 4.2 μL of 4 mM peptide reagent (5 equivalents to the antibody), and stir at room temperature for 1 hour. bottom. The reaction solution was added to a NAP-5 column to stop the reaction and replaced with 20 mM PBS buffer. When the mass was measured by ESI-TOFMS, a peak was observed at 148065 for the raw material trastuzumab, a peak was observed at 152525 for the product into which one binding peptide was introduced, and two binding peptides were introduced. A peak was observed at 156,978.

(39-5) HIC-UPLC Analysis of Specific Modification of Anti-HER2 IgG Antibody Trastuzumab The antibody/peptide complex produced in (39-4) and the starting antibody were analyzed by HIC. A Protein-Pak Hi Res HIC Column (Waters) 4.6×100 mm 2.5 μm was used as the column. A_Buffer: 0.1 M PiNa, 2.3 M (NH ₄ ) ₂ SO ₄ , pH 7.0, B_Buffer: 0.1 M PiNa, pH 7.0, flow rate 0.6 ml/min, gradient A60% B40%→A0 %
B100%, 16 min (20 min of data collection), column temperature of 40° C., thermostat temperature of 40° C., detector at wavelength of 280 nm.

The samples were reacted under the following conditions.
a: Trastuzumab raw material b: Trastuzumab + peptide disulfide linker conjugate-NHS activated form 5 equivalents;

Retension Time 9.2124 minutes is considered to be a compound into which one peptide was introduced, and 8.6157 minutes is considered to be a compound into which two peptides were introduced (Fig. 50).

[Example 40: Peptide mapping of thiol-introducer of trastuzumab]
(40-1) Trypsin treatment of trastuzumab thiol introducer 10 μL of sample solution, 50 mM ammonium bicarbonate buffer, 10 μL of 20 mM dithiothreitol aqueous solution dissolved in 40% trifluoroethanol in a 1.5 mL low-adsorption microtest tube. In addition, after heating at 65° C. for 1 hour, 10 μL of a 50 mM iodoacetamide aqueous solution was added, and the reaction was allowed to proceed by shaking at 300 rpm for 30 minutes at room temperature in the dark. After the reaction, 40 μL of 50 mM ammonium bicarbonate buffer was added and stirred, and 10 μL of 20 ng/μL trypsin aqueous solution (Proteomics Grade, Code No. T6567-5×20 μg (SIGMA)) was added for enzymatic digestion at 37° C. for 18 hours. After digestion, 2 μL of 20% trifluoroacetic acid aqueous solution was added to stop the reaction, and LC-MS/MS measurement was performed.

(40-2) Trastuzumab LC-MS/MS measurement conditions (analytical device)
Nano HPLC: EASY-nLC 1000 (Thermo Fisher Scientific)
Mass spectrometer: Tribrid mass spectrometer Orbitrap Fusion (Thermo Fisher Scientific)

(HPLC analysis conditions)
Trap column: Acclaim PepMap® 100, 75 μm x 2 cm (Thermo Fisher Scientific)
Analysis column: ESI-column (NTCC-360/75-3-125, 75 μm×12.5 cm, 3 μm (Nikkyo Technos Co., Ltd.))
Mobile phase A: 0.1% formic acid aqueous solution Mobile phase B: 0.1% formic acid, acetonitrile solution Loading solution: 0.1% trifluoroacetic acid aqueous solution Flow rate: 300 nL/min
Sample injection volume: 1 μL
Gradient conditions (B%): 2% (0.0-0.5 minutes), 2% → 30% (0.5-23.5 minutes), 30% → 75% (23.5-25.5 minutes ), 75% (25.5-35.0 min)

(Mass spectrometer analysis conditions)
Ionization method: ESI, Positive mode Scan type: Data Dependent Aquisition
Activation Type: Collision Induced Dissociation (CID)
Acquisition of data was carried out using attached software Xcalibur 3.0 (Thermo Fisher Scientific) and Thermo Orbitrap Fusion Tune Application 2.0 (Thermo Fisher Scientific).

(40-3) Analysis Conditions for Modified Site of Trastuzumab Modified site analysis for LC-MS/MS measurement results was performed using Proteome Discoverer version 1.4 (Thermo Fisher Scientific).

The analysis with Proteome Discoverer used Sequest HT as a search engine, with a precursor ion range of 350-5000 Da and a Total Intensity Threshold of 50000. Also, trypsin was set as a digestive enzyme, and Maximum Missed Cleavage Sites was set to 3. Also, the mass tolerances of precursor and fragment ions were set to 5 ppm and 0.5 Da, respectively. Carbamidomethyl (+57.021 Da) was set as Static Modification as modification of cysteine residue with iodoacetamide. Dynamic Modifications were set for oxidation of methionine (+15.995 Da) and modification to lysine residues (thiol introduction (+145.019 Da) after carbamidomethylation with iodoacetamide). Furthermore, a filter was applied so that Peptide Confidence was High only.

In addition, (1) and (3) shown in FIG. 8 were used as the amino acid sequence data to be searched for the modification site.

(40-4) Trastuzumab modification site analysis results by LC-MS/MS As a result of analysis using LC-MS/MS, trastuzumab modified with the binder peptide of Example 39 was converted to a lysine residue by tryptic digestion. MS spectrum of a peptide fragment of VVSVLTVLHQDWLNGKEYK (SEQ ID NO: 66), a peptide consisting of 19 amino acid residues containing a modified site (thiol-introduced with iodoacetamide (+145.019 Da)) (obtained value: m / z 791.74872, theoretical value 791.74753, trivalent) was observed (Fig. 51), and the m/ A product ion with z 1114.35 (theoretical value: 1114.06) was confirmed (Fig. 52). In addition, a peptide fragment of EEQYDSTYRVVSVLTVLHQDWLNGKEYK (SEQ ID NO: 67), a peptide consisting of 28 amino acid residues containing a modification site to a lysine residue by trypsin digestion (a thiol introduction (+145.019 Da) subjected to carbamidomethylation with iodoacetamide), was obtained. MS spectrum (actual value: m / z 886.93408, theoretical value: 886.93215, tetravalent) was observed (Fig. 53), and the CID spectrum also shows modification of the lysine residue at position 317 of the heavy chain, 2 A product ion with m/z 938.70 (theoretical value: 938.46) corresponding to the valence y14 was confirmed (Fig. 54).

[Example 41: Synthesis of a compound (peptide disulfide linker conjugate-NHS activator) having an affinity substance for soluble proteins, a cleavable moiety and a reactive group, and modification of the anti-HER2 IgG antibody trastuzumab using the compound and its analysis]
(41-1) Synthesis of IgG1 Fc-Binding Peptide The sequence of Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC-NH ₂ (SEQ ID NO: 68) was synthesized in the same manner as in Example 1 by the Fmoc solid-phase synthesis method. got the target.

上記アミノ酸配列は、配列番号６８である。 (41-2) Formation of intramolecular disulfide bond at Cys at positions 5 and 34 of Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC-NH ₂ (SEQ ID NO: 68)

The amino acid sequence is SEQ ID NO:68.

The peptide synthesized in (41-1) was dissolved in DMSO, and 0.1M Tris-HCl pH 8.0 was added. Glutathione oxidized form was added to this solution and stirred at room temperature for 20 hours. A 2M trifluoroacetic acid aqueous solution was added to the reaction solution to stop the reaction, and the solution was dissolved in a 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. , eluted with a mixed solution of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain the desired product (20.0 mg, 4.70 μmol).

MS (ESI) m/z: z = 4 1063.65 [M+4H] ⁴⁺ , z = 5 851.15 [M+5H] ⁵⁺

上記アミノ酸配列は、配列番号６８である。 (41-3) Linking disulfide linker and peptide

The amino acid sequence is SEQ ID NO:68.

Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC-NH ₂ (SEQ ID NO: 68) synthesized in (41-2) (20.0 mg, 4.70 μmol, provided that two cysteines at positions 5 and 34 each form an intramolecular disulfide bond ) was dissolved in N,N-dimethylformamide (1.00 mL), and di(N-succinimidyl) 3,3′-dithiodipropionate (76.0 mg, 188 μmol) was dissolved in acetonitrile (1.00 mL). was added and stirred at room temperature for 24 hours. After removing acetonitrile by concentrating under reduced pressure, this was dissolved in 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. Elution was performed with a mixed solution of water and acetonitrile containing 0.05% acetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain the peptide disulfide linker conjugate-NHS activated product (12.0 mg, 2.63 μmol). .

MS (ESI) m/z: z = 5 908.95 [M+5H] ⁵⁺ , z = 6 757.75 [M+6H] ⁶⁺
HPLC Purity: 83%

(41-4) Specific modification of anti-HER2 IgG antibody trastuzumab and analysis by ESI-TOFMS The peptide disulfide linker conjugate-NHS-activated product synthesized in (41-3) was dissolved in N,N'-dimethylformamide and bottom. Dissolve 500 µg of anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical Co., Ltd.) in 171 µL of 50 mM sodium acetate buffer (pH 5.5), add 8.5 µL of 4 mM peptide reagent (10 equivalents to the antibody), and stir at room temperature for 1 hour. bottom. The reaction solution was added to a NAP-5 column to stop the reaction and replaced with 20 mM PBS buffer. When the mass was measured by ESI-TOFMS, a peak was observed at 148234 for the raw material trastuzumab, a peak was observed at 152648 for the product into which one binding peptide was introduced, and two binding peptides were introduced. A peak was observed at 157,083.

(41-5) Confirmation of heavy chain selectivity by ESI-TOFMS analysis under reducing conditions of a specific modification of trastuzumab 7 mM tris(2-carboxyethyl)phosphine on the antibody/peptide complex produced in (41-4) 5 μL of hydrochloride solution (equal volume to antibody) was added and stirred at room temperature for 20 minutes. When the mass was measured by ESI-TOFMS, light chain peaks were observed at 50,686 and 50,847 in which a thiopropionyl group was introduced into the heavy chain of the product, and at 23,439, which was the same as the raw material.

(41-6) HIC-UPLC Analysis of Specific Modification of Anti-HER2 IgG Antibody Trastuzumab The antibody/peptide complex produced in (41-4) and the starting antibody were analyzed by HIC. A Protein-Pak Hi Res HIC Column (Waters) 4.6×100 mm 2.5 μm was used as the column. A_Buffer: 0.1 M PiNa, 2.3 M (NH ₄ ) ₂ SO ₄ , pH 7.0, B_Buffer: 0.1 M PiNa, pH 7.0, flow rate 0.6 ml/min, gradient A60% B40%→A0 %
B100%, 16 min (20 min of data collection), column temperature of 40° C., thermostat temperature of 40° C., detector at wavelength of 280 nm.

The samples were reacted under the following conditions.
a: Trastuzumab raw material b: Trastuzumab + peptide disulfide linker conjugate-NHS activated form 10 equivalents;

The retention time of 9.7202 minutes is considered to be the trastuzumab raw material, 9.2689 minutes to the compound into which one peptide was introduced, and 8.7994 minutes to the compound into which two peptides were introduced (Fig. 55).

[Example 42: Peptide mapping of thiol-introducer of trastuzumab]
(42-1) Trastuzumab thiol derivative was treated with trypsin in the same manner as (40-1).

(42-2) The same measurement conditions as the LC-MS/MS measurement conditions for trastuzumab (40-2) were used.

(42-3) Analysis conditions for modified site of trastuzumab The same analysis conditions as (40-3) were used.

(42-4) Results of Analysis of Modified Sites of Trastuzumab by LC-MS/MS As a result of analysis using LC-MS/MS, trastuzumab modified with the binder peptide of Example 41 was converted to lysine residues by tryptic digestion. MS spectrum of a peptide fragment of FNWYVDGVEVHNAKTKPR (SEQ ID NO: 69), a peptide consisting of 18 amino acid residues containing a modified site (thiol-introduced with iodoacetamide (+145.019 Da)) (obtained value: m / z 769.04688, theoretical value: 769.04457, trivalent) was observed (Fig. 56), and the CID spectrum indicates modification of the lysine residue at position 288 or 290 in EU numbering of the heavy chain, to divalent y12 The corresponding product ion with m/z 741.15 (theoretical value: 740.89) was confirmed (Fig. 57).

[Example 43: Synthesis of a compound (peptide disulfide linker conjugate-NHS activator) having an affinity substance for soluble proteins, a cleavable moiety and a reactive group, and modification of the anti-HER2 IgG antibody trastuzumab using the compound and its analysis]
(43-1) Synthesis of IgG1 Fc-Binding Peptide The sequence of Ac-FNKQCQRRFYEALHDPNLNEEQRNARIRSIRDDC-NH ₂ (SEQ ID NO: 70) was synthesized in the same manner as in Example 1 by the Fmoc solid-phase synthesis method. got the target.

上記アミノ酸配列は、配列番号７０である。 (43-2) Formation of intramolecular disulfide bond at Cys at positions 5 and 34 of Ac-FNKQCQRRFYEALHDPNLNEEQRNARIRSIRDDC-NH ₂ (SEQ ID NO: 70)

The amino acid sequence is SEQ ID NO:70.

The peptide synthesized in (43-1) was dissolved in DMSO, and 0.1M Tris-HCl pH 8.0 was added. Glutathione oxidized form was added to this solution and stirred at room temperature for 20 hours. A 2M trifluoroacetic acid aqueous solution was added to the reaction solution to stop the reaction, and the solution was dissolved in a 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. , eluted with a mixed solution of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain the desired product (20.0 mg, 4.67 μmol).

MS (ESI) m/z: z = 5 848.30 [M+5H] ⁵⁺ , z = 6 707.10 [M+6H] ⁶⁺

上記アミノ酸配列は、配列番号７０である。 (43-3) Linking disulfide linker and peptide

The amino acid sequence is SEQ ID NO:70.

Ac-FNKQCQRRFYEALHDPNLNEEQRNARIRSIRDDC-NH ₂ (SEQ ID NO: 70) synthesized in (43-2) (20.0 mg, 4.67 μmol, provided that two cysteines at positions 5 and 34 each form an intramolecular disulfide bond) ) was dissolved in N,N-dimethylformamide (0.50 mL), and di(N-succinimidyl) 3,3′-dithiodipropionate (76.0 mg, 188 μmol) was dissolved in acetonitrile (0.60 mL). was added and stirred at room temperature for 24 hours. After removing acetonitrile by concentrating under reduced pressure, this was dissolved in 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. Elution was performed with a mixed solution of water and acetonitrile containing 0.05% acetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain the peptide disulfide linker conjugate-NHS activated product (9.00 mg, 1.97 μmol). .

MS (ESI) m/z: z=6 761.85 [M+6H] ⁶⁺
HPLC Purity: 85%

(43-4) Specific modification of anti-HER2 IgG antibody trastuzumab and analysis by ESI-TOFMS The peptide disulfide linker conjugate-NHS-activated product synthesized in (43-3) was dissolved in N,N'-dimethylformamide and bottom. 1000 µg of the anti-HER2 IgG antibody trastuzumab was dissolved in 342 µL of 50 mM sodium acetate buffer (pH 5.5), 17.0 µL of 4 mM peptide reagent (10 equivalents to the antibody) was added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was replaced with 100 mM sodium citrate buffer (pH 2.9) to stop the reaction, and further replaced with 20 mM PBS buffer. When the mass was measured by ESI-TOFMS, a peak at 156963 was confirmed for a product into which two binding peptides had been introduced.

(43-5) Confirmation of heavy chain selectivity by ESI-TOFMS analysis under reducing conditions of a specific modification of trastuzumab 7 mM tris(2-carboxyethyl)phosphine on the antibody/peptide complex produced in (43-4) 5 μL of hydrochloride solution (equal volume to antibody) was added and stirred at room temperature for 20 minutes. When the mass was measured by ESI-TOFMS, light chain peaks were observed at 50,686 and 50,847 in which a thiopropionyl group was introduced into the heavy chain of the product, and at 23,439, which was the same as the raw material.

(43-6) HIC-UPLC Analysis of Specific Modification of Anti-HER2 IgG Antibody Trastuzumab The antibody/peptide complex produced in (43-4) and the starting antibody were analyzed by HIC. A Protein-Pak Hi Res HIC Column (Waters) 4.6×100 mm 2.5 μm was used as the column. A_Buffer: 0.1 M PiNa, 2.3 M (NH ₄ ) ₂ SO ₄ , pH 7.0, B_Buffer: 0.1 M PiNa, pH 7.0, flow rate 0.6 ml/min, gradient A60% B40%→A0 %
B100%, 16 min (20 min of data collection), column temperature of 40° C., thermostat temperature of 40° C., detector at wavelength of 280 nm.

The samples were reacted under the following conditions.
a: Trastuzumab raw material b: Trastuzumab + peptide disulfide linker conjugate-NHS activated form 10 equivalents;

Retension Time 8.6251 minutes is considered to be a compound into which two peptides were introduced (Fig. 58).

[Example 44: Peptide mapping of thiol-introducer of trastuzumab]
(44-1) Trypsin treatment of thiol introducer of trastuzumab 10 μL of sample solution, 50 mM ammonium bicarbonate buffer, 10 μL of 20 mM dithiothreitol aqueous solution dissolved in 40% trifluoroethanol in a 1.5 mL low-adsorption microtest tube. In addition, after heating at 65° C. for 1 hour, 10 μL of a 50 mM iodoacetamide aqueous solution was added, and the reaction was allowed to proceed by shaking at 300 rpm for 30 minutes at room temperature in the dark. After the reaction, 40 μL of 50 mM ammonium hydrogen carbonate buffer was added and stirred, 10 μL of 20 ng/μL trypsin aqueous solution was added, and enzymatic digestion was performed at 37° C. for 18 hours. After digestion, 2 μL of 20% trifluoroacetic acid aqueous solution was added to stop the reaction. Then, it was diluted 10-fold with 50 mM ammonium bicarbonate buffer and 0.5% trifluoroacetic acid and used for LC-MS/MS measurement.

(44-2) Trastuzumab LC-MS/MS measurement conditions (analytical device)
Nano HPLC: EASY-nLC 1000 (Thermo Fisher Scientific)
Mass spectrometer: Tribrid mass spectrometer Orbitrap Fusion (Thermo Fisher Scientific)

(HPLC analysis conditions)
Trap column: Acclaim PepMap® 100, 75 μm x 2 cm (Thermo Fisher Scientific)
Analysis column: ESI-column (NTCC-360/75-3-125, 75 μm×12.5 cm, 3 μm (Nikkyo Technos Co., Ltd.))
Mobile phase A: 0.1% formic acid aqueous solution Mobile phase B: 0.1% formic acid, acetonitrile solution Loading solution: 0.1% trifluoroacetic acid aqueous solution Flow rate: 300 nL/min
Sample injection volume: 1 μL
Gradient conditions (B%): 2% (0.0-0.5 minutes), 2% → 30% (0.5-23.5 minutes), 30% → 75% (23.5-25.5 minutes ), 75% (25.5-35.0 min)

(Mass spectrometer analysis conditions)
Ionization method: ESI, Positive mode Scan type: Data Dependent Aquisition
Activation Type: Collision Induced Dissociation (CID)
Acquisition of data was carried out using attached software Xcalibur 3.0 (Thermo Fisher Scientific) and Thermo Orbitrap Fusion Tune Application 2.0 (Thermo Fisher Scientific).

(44-3) Analysis Conditions for Modified Site of Trastuzumab Modified site analysis for LC-MS/MS measurement results was performed using Proteome Discoverer version 1.4 (Thermo Fisher Scientific).

Analyzes on Proteome Discoverer used Sequest HT as the search engine with a precursor ion range of 350-5000 Da. In addition, trypsin is set as a digestive enzyme and Maximum Missed Cleavage
Sites was set to 3. Also, the mass tolerances of precursor and fragment ions were set to 5 ppm and 0.5 Da, respectively. Carbamidomethyl (+57.021 Da) was set as Static Modification as modification of cysteine residue with iodoacetamide. Dynamic Modifications were set for oxidation of methionine (+15.995 Da) and modification to lysine residues (thiol introduction (+145.019 Da) after carbamidomethylation with iodoacetamide). Furthermore, a filter was applied so that Peptide Confidence was High only. The Total Intensity Threshold was set to 32000 only for Example 43.

In addition, (1) and (3) shown in FIG. 8 were used as the amino acid sequence data to be searched for the modification site.

(44-4) Results of analysis of modified site of trastuzumab by LC-MS/MS As a result of analysis using LC-MS/MS, the modified site of lysine residue by tryptic digestion of trastuzumab (carbamidomethylated by iodoacetamide) MS spectrum of a peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 40) consisting of 33 amino acid residues containing a thiol derivative (+145.019 Da)) (actual value: m / z 952.23145, theoretical value: 952.22900, tetravalent) was observed (Fig. 59), and m/z 968.30 (theoretical value: 968. 01) was confirmed (Fig. 60).

[Example 45: Synthesis of a compound (peptide disulfide linker conjugate-NHS activator) having an affinity substance for soluble proteins, a cleavable moiety and a reactive group, and modification of the anti-HER2 IgG antibody trastuzumab using the compound and its analysis]
(45-1) Synthesis of IgG1 Fc-Binding Peptide The sequence of Ac-FNMQCKRRFYEALHDPNLNEEQRNARIRSIRDDC-NH ₂ (SEQ ID NO: 71) was synthesized in the same manner as in Example 1 by the Fmoc solid-phase synthesis method. got the target.

上記アミノ酸配列は、配列番号７１である。 (45-2) Formation of intramolecular disulfide bond at Cys at positions 5 and 34 of Ac-FNMQCKRRFYEALHDPNLNEEQRNARIRSIRDDC-NH ₂ (SEQ ID NO: 71)

The amino acid sequence is SEQ ID NO:71.

The peptide synthesized in (45-1) was dissolved in DMSO, and 0.1M Tris-HCl pH 8.0 was added. Glutathione oxidized form was added to this solution and stirred at room temperature for 20 hours. A 2M trifluoroacetic acid aqueous solution was added to the reaction solution to stop the reaction, and the solution was dissolved in a 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. , eluted with a mixed solution of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain the desired product (20.0 mg, 4.67 μmol).

MS (ESI) m/z: z=3 692.70 [M+3H] ^<3+> , z=4 519.80 [M+4H] ^<4+>

上記アミノ酸配列は、配列番号７１である。 (45-3) Linking disulfide linker and peptide

The amino acid sequence is SEQ ID NO:71.

Ac-FNMQCKRRFYEALHDPNLNEEQRNARIRSIRDDC-NH ₂ (SEQ ID NO: 71) synthesized in (45-2) (20.0 mg, 4.67 μmol, provided that two cysteines at positions 5 and 34 each form an intramolecular disulfide bond) ) was dissolved in N,N-dimethylformamide (0.50 mL), and di(N-succinimidyl) 3,3′-dithiodipropionate (76.0 mg, 188 μmol) was dissolved in acetonitrile (0.60 mL). was added and stirred at room temperature for 24 hours. After removing acetonitrile by concentrating under reduced pressure, this was dissolved in 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. Elution was performed with a mixed solution of water and acetonitrile containing 0.05% acetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain the peptide disulfide linker conjugate-NHS activated form (5.50 mg, 1.20 μmol). .

MS (ESI) m/z: z=6 762.40 [M+6H] ⁶⁺
HPLC Purity: 100%

(45-3) Specific modification of anti-HER2 IgG antibody trastuzumab and analysis by ESI-TOFMS The peptide disulfide linker conjugate-NHS-activated product synthesized in (45-2) was dissolved in N,N'-dimethylformamide and bottom. 500 µg of the anti-HER2 IgG antibody trastuzumab was dissolved in 171 µL of 50 mM sodium acetate buffer (pH 5.5), 8.5 µL of 4 mM peptide reagent (10 equivalents to the antibody) was added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was replaced with 100 mM sodium citrate buffer (pH 2.9) to stop the reaction, and further replaced with 20 mM PBS buffer. When the mass was measured by ESI-TOFMS, a peak was observed at 148226 for the raw material trastuzumab, and a peak at 152662 was confirmed for the product into which one binding peptide had been introduced.

(45-4) Confirmation of heavy chain selectivity by ESI-TOFMS analysis under reducing conditions of a specific modification of trastuzumab 7 mM tris(2-carboxyethyl)phosphine on the antibody/peptide complex produced in (45-3) 5 μL of hydrochloride solution (equal volume to antibody) was added and stirred at room temperature for 20 minutes. When the mass was measured by ESI-TOFMS, light chain peaks were observed at 50,686 and 50,847 in which a thiopropionyl group was introduced into the heavy chain of the product, and at 23,439, which was the same as the raw material.

(45-5) HIC-UPLC Analysis of Specific Modification of Anti-HER2 IgG Antibody Trastuzumab The antibody/peptide complex produced in (45-3) and the starting antibody were analyzed by HIC. A Protein-Pak Hi Res HIC Column (Waters) 4.6×100 mm 2.5 μm was used as the column. A_Buffer: 0.1 M PiNa, 2.3 M (NH ₄ ) ₂ SO ₄ , pH 7.0, B_Buffer: 0.1 M PiNa, pH 7.0, flow rate 0.6 ml/min, gradient A60% B40%→A0 %
B100%, 16 min (20 min of data collection), column temperature of 40° C., thermostat temperature of 40° C., detector at wavelength of 280 nm.

The samples were reacted under the following conditions.
a: Trastuzumab raw material b: Trastuzumab + peptide disulfide linker conjugate-NHS activated form 10 equivalents;

Retension Time 9.8954 minutes is considered to be a trastuzumab raw material, and 9.4892 minutes is considered to be a compound in which one peptide is introduced into trastuzumab (Fig. 61).

[Example 46: Peptide mapping of thiol-introducer of trastuzumab]
(46-1) Trypsin treatment of thiol-introducer of trastuzumab The same treatment as in (44-1) was performed.

(46-2) Trastuzumab LC-MS/MS measurement conditions The same measurement conditions as in (44-2) were used.

(46-3) Analysis Conditions for Modified Site of Trastuzumab The same analysis conditions as in (44-3) were used.

(46-4) Results of modification site analysis of trastuzumab by LC-MS/MS As a result of analysis using LC-MS/MS, the site of modification of lysine residues by trypsin digestion of trastuzumab (carbamidomethylation by iodoacetamide) MS spectrum of a peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 40) consisting of 33 amino acid residues containing a thiol derivative (+145.019 Da)) (actual value: m / z 952.22968, theoretical value: 952.22900, tetravalent) was observed (Fig. 62), and m/z 764.60 (theoretical value: 764.60) corresponding to bivalent y12, which indicates modification of the lysine residue at position 246 in EU numbering of the heavy chain from the CID spectrum. 40) was confirmed (Fig. 63).

[Example 47: Synthesis of a compound (peptide disulfide linker conjugate-NHS activator) having an affinity substance for soluble proteins, a cleavable moiety and a reactive group, and modification of the anti-HER2 IgG antibody trastuzumab using the compound and its analysis]
(47-1) Synthesis of IgG1 Fc-Binding Peptide The sequence of Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIRKDC-NH ₂ (SEQ ID NO: 72) was synthesized in the same manner as in Example 1 by the Fmoc solid-phase synthesis method. got the target.

上記アミノ酸配列は、配列番号７２である。 (47-2) Formation of intramolecular disulfide bond at Cys at positions 5 and 34 of Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIRKDC-NH ₂ (SEQ ID NO: 72)

The amino acid sequence is SEQ ID NO:72.

The peptide synthesized in (47-1) was dissolved in DMSO, and 0.1M Tris-HCl pH 8.0 was added. Glutathione oxidized form was added to this solution and stirred at room temperature for 20 hours. A 2M trifluoroacetic acid aqueous solution was added to the reaction solution to stop the reaction, and the solution was dissolved in a 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. , eluted with a mixed solution of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and freeze-dried to obtain the desired product (24.5 mg, 5.71 μmol).

MS (ESI) m/z: z=3 692.70 [M+3H] ^<3+> , z=4 519.80 [M+4H] ^<4+>

上記アミノ酸配列は、配列番号７２である。 (47-3) Linking disulfide linker and peptide

The amino acid sequence is SEQ ID NO:72.

Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIRKDC-NH ₂ (SEQ ID NO: 72) synthesized in (47-2) (24.5 mg, 5.71 μmol, provided that two cysteines at positions 5 and 34 each form an intramolecular disulfide bond) ) was dissolved in N,N-dimethylformamide (1.00 mL), and di(N-succinimidyl) 3,3′-dithiodipropionate (92.2 mg, 228 μmol) was dissolved in acetonitrile (1.00 mL). was added and stirred at room temperature for 24 hours. After removing acetonitrile by concentrating under reduced pressure, this was dissolved in 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing material. Elution was performed with a mixed solution of water and acetonitrile containing 0.05% acetic acid, and each fraction was confirmed by LC-MS. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain the peptide disulfide linker conjugate-NHS activated product (7.50 mg, 1.64 μmol). .

MS (ESI) m/z: z=6 762.40 [M+6H] ⁶⁺
HPLC Purity: 84%

(47-4) Specific modification of anti-HER2 IgG antibody trastuzumab and analysis by ESI-TOFMS The peptide disulfide linker conjugate-NHS-activated product synthesized in (47-3) was dissolved in N,N'-dimethylformamide and bottom. 500 µg of the anti-HER2 IgG antibody trastuzumab was dissolved in 171 µL of 50 mM sodium acetate buffer (pH 5.5), 8.5 µL of 4 mM peptide reagent (10 equivalents to the antibody) was added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was replaced with 100 mM sodium citrate buffer (pH 2.9) to stop the reaction, and further replaced with 20 mM PBS buffer. When the mass was measured by ESI-TOFMS, a peak was observed at 148087 for the raw material trastuzumab, and a peak was confirmed at 152694 for the product into which one binding peptide was introduced. A peak was confirmed at 157,160.

(47-5) Confirmation of heavy chain selectivity by ESI-TOFMS analysis under reducing conditions of a specific modification of trastuzumab 7 mM tris(2-carboxyethyl)phosphine on the antibody/peptide complex produced in (47-4) 5 μL of hydrochloride solution (equal volume to antibody) was added and stirred at room temperature for 20 minutes. When the mass was measured by ESI-TOFMS, light chain peaks were observed at 50,686 and 50,847 in which a thiopropionyl group was introduced into the heavy chain of the product, and at 23,439, which was the same as the raw material.

(47-6) HIC-UPLC Analysis of Specific Modification of Anti-HER2 IgG Antibody Trastuzumab The antibody/peptide complex produced in (47-4) and the starting antibody were analyzed by HIC. A Protein-Pak Hi Res HIC Column (Waters) 4.6×100 mm 2.5 μm was used as the column. A_Buffer: 0.1 M PiNa, 2.3 M (NH ₄ ) ₂ SO ₄ , pH 7.0, B_Buffer: 0.1 M PiNa, pH 7.0, flow rate 0.6 ml/min, gradient A60% B40%→A0 %
B100%, 16 min (20 min of data collection), column temperature of 40° C., thermostat temperature of 40° C., detector at wavelength of 280 nm.

The samples were reacted under the following conditions.
a: Trastuzumab raw material b: Trastuzumab + peptide disulfide linker conjugate-NHS activated form 10 equivalents;

Retension Time 9.5796 minutes is considered to be a trastuzumab raw material, and 9.1199 minutes is considered to be a compound in which one peptide is introduced into trastuzumab (Fig. 64).

[Example 48: Peptide mapping of thiol-introducer of trastuzumab]
(48-1) Trastuzumab thiol derivative was treated with trypsin in the same manner as (44-1).

(48-2) The same measurement conditions as the LC-MS/MS measurement conditions for trastuzumab (44-2) were used.

(48-3) Analysis conditions for modified site of trastuzumab The same analysis conditions as (44-3) were used.

(48-4) LC-MS/MS analysis results of modification site of trastuzumab As a result of analysis using LC-MS/MS, the modification site of trastuzumab to a lysine residue by trypsin digestion (carbamidomethylation by iodoacetamide) MS spectrum of a peptide fragment of FNWYVDGVEVHNAKTKPR (SEQ ID NO: 69), a peptide consisting of 33 amino acid residues containing a thiol-introduced (+145.019 Da)) (actual value: m / z 769.04529, theoretical value: 769.04457, trivalent) was observed (Fig. 65), and m/z 549.11 (theoretical value: 548. 79) was confirmed (Fig. 66).

[Example 49: Modification of a human IgG2 antibody using a compound having an affinity for soluble proteins, a cleavable moiety and a compound having a reactive group (peptide disulfide linker conjugate-NHS activator) and its analysis]
(49-1) Specific modification of human IgG2 antibody and analysis by ESI-TOFMS The peptide disulfide linker conjugate-NHS-activated product synthesized in Example (2-2) was dissolved in N,N'-dimethylformamide and dissolved at 4 mM. and Dissolve 500 μg of human IgG2 antibody (manufactured by RayBiotech) in 171 μL of 50 mM sodium acetate buffer (pH 5.5), add 8.5 μL of 4 mM peptide reagent (10 equivalents to the antibody), and stir at room temperature for 1 hour. bottom. The reaction solution was replaced with 100 mM sodium citrate buffer (pH 2.9) to stop the reaction, and further replaced with 20 mM PBS buffer. When the mass was measured by ESI-TOFMS, a peak was observed at 153047 for the starting IgG2 antibody, and a peak was confirmed at 155129 for the product into which one binding peptide had been introduced, indicating that two binding peptides had been introduced. A peak was confirmed at 157,699 for the product.

(49-2) HIC-UPLC Analysis of Specific Modification of Human IgG2 Antibody The antibody/peptide complex produced in (49-1) and the starting antibody were analyzed by HIC. A Protein-Pak Hi Res HIC Column (Waters) 4.6×100 mm 2.5 μm was used as the column. A_Buffer: 0.1 M PiNa, 2.3 M (NH ₄ ) ₂ SO ₄ , pH 7.0, B_Buffer: 0.1 M PiNa, pH 7.0, flow rate 0.6 ml/min, gradient A60% B40%→A0 %
B100%, 16 min (20 min of data collection), column temperature of 40° C., thermostat temperature of 40° C., detector at wavelength of 280 nm.

The samples were reacted under the following conditions.
a: Human IgG2 antibody raw material b: Human IgG2 antibody + peptide disulfide linker conjugate-NHS activator 10 equivalents;

Retension Time 9.0209 minutes is considered to be a human IgG2 antibody raw material, and 10.2043 minutes is considered to be a compound in which one peptide is introduced into a human IgG2 antibody (Fig. 67).

[Example 50: Modification of a human IgG2 antibody using a compound having an affinity for a soluble protein, a cleavable moiety, and a reactive group (peptide disulfide linker conjugate-NHS activator) and its analysis]
(50-1) Specific modification of human IgG2 antibody and analysis by ESI-TOFMS The peptide disulfide linker conjugate-NHS-activated product synthesized in Example (43-3) was dissolved in N,N'-dimethylformamide and dissolved at 4 mM. and Dissolve 500 μg of human IgG2 antibody (manufactured by RayBiotech) in 171 μL of 50 mM sodium acetate buffer (pH 5.5), add 8.5 μL of 4 mM peptide reagent (10 equivalents to the antibody), and stir at room temperature for 1 hour. bottom. 100 mM sodium citrate buffer (pH
2.9) to stop the reaction, and then replaced with 20 mM PBS buffer. When the mass was measured by ESI-TOFMS, a peak was observed at 153047 for the starting IgG2 antibody, and a peak was confirmed at 157652 for the product into which one binding peptide had been introduced, indicating that two binding peptides had been introduced. A peak was confirmed at 165,043 for the product.

(50-2) HIC-UPLC Analysis of Specific Modification of Human IgG2 Antibody The antibody/peptide complex produced in (50-1) and the starting antibody were analyzed by HIC. A Protein-Pak Hi Res HIC Column (Waters) 4.6×100 mm 2.5 μm was used as the column. A_Buffer: 0.1 M PiNa, 2.3 M (NH ₄ ) ₂ SO ₄ , pH 7.0, B_Buffer: 0.1 M PiNa, pH 7.0, flow rate 0.6 ml/min, gradient A60% B40%→A0 %
B100%, 16 min (20 min of data collection), column temperature of 40° C., thermostat temperature of 40° C., detector at wavelength of 280 nm.

The samples were reacted under the following conditions.
a: Human IgG2 antibody raw material b: Human IgG2 antibody + peptide disulfide linker conjugate-NHS activator 10 equivalents;

Retension Time 9.0209 minutes is considered to be a human IgG2 antibody raw material, and 8.3882 minutes is considered to be a compound in which one peptide is introduced into a human IgG2 antibody (Fig. 68).

[Example 51: Modification of a human IgG4 antibody using a compound having an affinity substance for a soluble protein, a cleavable moiety and a reactive group (peptide disulfide linker conjugate-NHS activator) and its analysis]
(51-1) Specific modification of human IgG4 antibody and analysis by ESI-TOFMS The peptide disulfide linker conjugate-NHS-activated product synthesized in Example (2-2) was dissolved in N,N'-dimethylformamide and dissolved at 4 mM. and Dissolve 500 µg of human IgG4 antibody (manufactured by RayBiotech) in 171 µL of 50 mM sodium acetate buffer (pH 5.5), add 8.5 µL of 4 mM peptide reagent (10 equivalents to the antibody), and stir at room temperature for 1 hour. bottom. The reaction solution was replaced with 100 mM sodium citrate buffer (pH 2.9) to stop the reaction, and further replaced with 20 mM PBS buffer. When the mass was measured by ESI-TOFMS, a peak was observed at 154618 for the starting IgG2 antibody and a peak at 159130 for the product into which two binding peptides were introduced.

(51-2) HIC-UPLC Analysis of Specific Modification of Human IgG4 Antibody The antibody/peptide complex produced in (51-1) and the starting antibody were analyzed by HIC. A Protein-Pak Hi Res HIC Column (Waters) 4.6×100 mm 2.5 μm was used as the column. A_Buffer: 0.1 M PiNa, 2.3 M (NH ₄ ) ₂ SO ₄ , pH 7.0, B_Buffer: 0.1 M PiNa, pH 7.0, flow rate 0.6 ml/min, gradient A60% B40%→A0 %
B100%, 16 min (20 min of data collection), column temperature of 40° C., thermostat temperature of 40° C., detector at wavelength of 280 nm.

The samples were reacted under the following conditions.
a: human IgG4 antibody raw material b: human IgG4 antibody + peptide disulfide linker conjugate-NHS activated form 10 equivalents;

Retension Time 10.3154 minutes is considered to be a human IgG4 antibody raw material, and 11.2450 minutes is considered to be a compound in which one peptide is introduced into a human IgG4 antibody (Fig. 69).

[Example 52: Modification of a human IgG4 antibody using a compound having an affinity for soluble proteins, a cleavable moiety and a compound having a reactive group (peptide disulfide linker conjugate-NHS activator) and its analysis]
(52-1) Specific modification of human IgG4 antibody and analysis by ESI-TOFMS The peptide disulfide linker conjugate-NHS-activated product synthesized in Example (43-3) was dissolved in N,N'-dimethylformamide and dissolved at 4 mM. and Dissolve 500 μg of human IgG2 antibody (manufactured by RayBiotech) in 171 μL of 50 mM sodium acetate buffer (pH 5.5), add 8.5 μL of 4 mM peptide reagent (10 equivalents to the antibody), and stir at room temperature for 1 hour. bottom. 100 mM sodium citrate buffer (pH
2.9) to stop the reaction, and then replaced with 20 mM PBS buffer. When the mass was measured by ESI-TOFMS, a peak was observed at 154,618 for the starting IgG2 antibody, and a peak at 163,530 for the product into which two binding peptides had been introduced was confirmed.

(52-2) HIC-UPLC Analysis of Specific Modification of Human IgG4 Antibody The antibody/peptide complex produced in (52-1) and the starting antibody were analyzed by HIC. A Protein-Pak Hi Res HIC Column (Waters) 4.6×100 mm 2.5 μm was used as the column. A_Buffer: 0.1 M PiNa, 2.3 M (NH ₄ ) ₂ SO ₄ , pH 7.0, B_Buffer: 0.1 M PiNa, pH 7.0, flow rate 0.6 ml/min, gradient A60% B40%→A0 %
B100%, 16 min (20 min of data collection), column temperature of 40° C., thermostat temperature of 40° C., detector at wavelength of 280 nm.

The samples were reacted under the following conditions.
a: human IgG4 antibody raw material b: human IgG4 antibody + peptide disulfide linker conjugate-NHS activated form 10 equivalents;

Retension Time 10.3154 minutes is considered to be a human IgG4 antibody raw material, and 8.8645 minutes is a compound in which one peptide is introduced into a human IgG4 antibody (Fig. 7
0).

[Example 53: (1) Synthesis of a cleavable linker and (2) Linking the synthesized linker to an IgG1 Fc-binding peptide, a compound having an affinity substance for a soluble protein, a cleavable moiety and a reactive group ( Synthesis of peptide-azido-modified thioester linker conjugate-NHS-activated product)]
(53-1) Synthesis of thioester linker and linking with peptide (53-1-1) Synthesis of thioester linker

5-Azidopentanoic acid (200 mg, 1.40 mmol) was dissolved in THF solvent (12 mL), and pivaloyl chloride (206 μL, 1.68 mmol) and N-methylmorpholine (230 μL, 2.10 mmol) were added at 0°C. The mixture was added and stirred for 30 minutes to prepare a mixed acid anhydride of 5-azidopentanoic acid. After dissolving 4-(tert-butoxy-oxo)-2-amino-butanoic acid (344 mg, 1.82 mmol) in 1 M aqueous sodium hydroxide solution (1.82 mL) at room temperature, the above 5-azidopentanoic acid of the mixed acid anhydride in THF was added dropwise at room temperature. After stirring at room temperature for 16 hours, a 6M hydrochloric acid aqueous solution was added to adjust the pH of the system to 3.0. The reaction mixture was diluted with ethyl acetate, washed with water and brine, added with anhydrous magnesium sulfate, and allowed to stand for 5 minutes. After removing magnesium sulfate by filtration and concentrating under reduced pressure to obtain a crude product, it was purified by column chromatography (dichloromethane:methanol=10:1). Fractions containing the product were collected and concentrated under reduced pressure to give 4-(tert-butoxy-oxo)-2-(5-azidopenta-1-oxo)amino-butanoic acid (320 mg, 1.02 mmol). Obtained.

MS (ESI) m/z: 337 [M+Na] ⁺

4-(tert-butoxy-oxo)-2-(5-azidopenta-1-oxo)amino-butanoic acid (154 mg, 0.490 mmol) was dissolved in THF (4.9 mL) and isobutyl chlorocarbonate was added at 0°C. (83.6 μL, 0.637 mmol) and N-methylmorpholine (108 μL, 0.980 mmol) were added and stirred for 30 minutes to prepare the corresponding mixed acid anhydride. Mercaptopropionic acid (128 μL, 1.47 mmol) and N,N-diisopropylethylamine (253 μL, 1.47 mmol) were added dropwise at room temperature, and the THF solution of the aforementioned mixed acid anhydride was added dropwise at room temperature. After stirring at room temperature for 16 hours, 1M sodium hydroxide aqueous solution was added to adjust the pH of the system to 9.0. The reaction solution was washed with water and ethyl acetate, and the aqueous phase was recovered. A 6M hydrochloric acid aqueous solution was added to the aqueous phase to adjust the pH of the system to 3.0, followed by liquid separation and extraction with ethyl acetate. placed. 3-[4-(tert-butoxy-oxo)-2-(5-azidopent-1-oxo)amino-1-oxo]sulfanyl-propanoic acid was obtained by removing magnesium sulfate by filtration and concentrating under reduced pressure. (108 mg, 0.268 mmol) was obtained.

MS (ESI) m/z: 403 [M+H] ⁺

上記アミノ酸配列は、配列番号３９である。 (53-1-2) Linking thioester linker and peptide

The amino acid sequence is SEQ ID NO:39.

The peptide (31.8 mg, 15.3 μmol) of Ac-RGNCAYHKGQLVWCTYH-NH2 (SEQ ID NO: 39) synthesized in Example 1-2, provided that the two homocysteines at positions 4 and 14 each have intramolecular disulfide bonds. ) was dissolved in 1 mL of N,N′-dimethylformamide and 3-[4-(tert-butoxy-oxo)-2-(5-azidopent-1-oxo)amino-1-oxo]- Sulfanyl-propanoic acid (61.7 mg, 0.153 mmol), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (29.4 mg, 0.153 mmol), 1-hydroxybenzotriazole (10.4 mg, 0.0766 mmol) was dissolved in 1 mL of DMF and added into the system. After stirring at room temperature for 2 hours, a 0.1% trifluoroacetic acid aqueous solution was added and the mixture was eluted by reverse phase preparative chromatography. Fractions containing the product were collected and concentrated under reduced pressure to remove only acetonitrile, followed by freeze-drying to obtain the peptide linker-linked-tBu form (2.5 mg, 1.02 μmol).

MS (ESI) m/z: z=2 1230 [M+2H] ^<2+> , z=3 820 [M+3H] ^<3+>

上記アミノ酸配列は、配列番号３９である。

The amino acid sequence is SEQ ID NO:39.

The above peptide linker conjugate (2.5 mg, 1.02 μmol) was dissolved in 0.250 mL of dichloromethane, 0.250 mL of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 1 hour. After concentrating under reduced pressure, 0.1% trifluoroacetic acid aqueous solution was added and elution was performed by reverse phase preparative chromatography. Fractions containing the product were collected and concentrated under reduced pressure to remove only acetonitrile, followed by freeze-drying to obtain the peptide linker conjugate-carboxylic acid form.

MS (ESI) m/z: z=2 1202[M+2H] ^<2+> , z=3 802[M+3H] ^<3+>

上記アミノ酸配列は、配列番号３９である。

The amino acid sequence is SEQ ID NO:39.

The peptide linker conjugate-carboxylic acid form was dissolved in 0.5 mL of N,N'-dimethylformamide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (2.0 mg, 10.2 μmol), N-Hydroxysuccinimide (1.2 mg, 10.2 μmol) was added and stirred for 16 hours. A 0.1% trifluoroacetic acid aqueous solution was added to the reaction mixture, and the mixture was eluted by reverse phase preparative chromatography. Fractions containing the product are collected and concentrated under reduced pressure to remove only acetonitrile, followed by freeze-drying to obtain the peptide-azido-modified thioester linker conjugate-NHS activated form (0.5 mg, 0.2 μmol). got

MS (ESI) m/z: z=2 1251 [M+2H] ^<2+> , z=3 834 [M+3H] ^<3+>

[Example 54: Specific modification of anti-HER2 IgG antibody trastuzumab and analysis by ESI-TOFMS]
(54-1) Specific modification of IgG antibody trastuzumab with azide-modified thioester linker-binding peptide reagent and analysis by ESI-TOFMS Peptide synthesized in (53-1-2) of Example 53-azido-modified thioester linker conjugate- The activated NHS was dissolved in N,N'-dimethylformamide to 4 mM. Dissolve 1.48 mg of anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical Co., Ltd.) in 555 μL of 60 mM sodium acetate buffer (pH 4.7), add 50 μL of 4 mM peptide reagent (20 equivalents to the antibody), and incubate at room temperature for 1 hour. The reaction was quenched with 100 mM citric acid by stirring. The reaction solution was solvent-exchanged with Amicon 10K to 9.57 mM PBS buffer (pH 7.4). When the mass was measured by ESI-TOFMS, a peak was observed at 148225 for the raw material trastuzumab, and a peak was observed at 153143 for the product into which two binding peptides were introduced.

(54-2) Confirmation of heavy chain selectivity by ESI-TOFMS analysis under reducing conditions of specific modification of anti-HER2 IgG antibody trastuzumab 2.0 μL of (2-carboxyethyl)phosphine hydrochloride solution (100 equivalents to the antibody) was added and stirred at room temperature for 20 minutes. When the mass was measured by ESI-TOFMS, the raw material trastuzumab had heavy chain peaks at 50595 and 50757, and a light chain peak at 23440. A light chain peak was observed at 23440, the same as the starting material.

(54-3) Cleavage of thioester linker and analysis by ESI-TOFMS The antibody/peptide thioester linker complex synthesized in (54-1) was dissolved in any buffer (pH 4.5 to pH 9.0), and hydroxylamine By adding an aqueous solution and stirring at room temperature, the thioester structure is hydrolyzed, leaving an azide group on the antibody. The mass of this can be measured by ESI-TOFMS.

(54-4) Linker Cleavage of Trastuzumab-Peptide Complex and ESI-TOFMS Analysis of Product Hydroxylamine, 9.56 mM PBS, and 4.8 g/mL EDTA were dissolved in pure water (pH 7.2) and stirred at room temperature to cleave the thioester bond in the linker. When the mass was measured by ESI-TOFMS, a peak was observed at 148469 where two azides were introduced into the product.

(54-5) Confirmation of heavy chain selectivity by ESI-TOFMS analysis under reducing conditions of a specific modification of trastuzumab 7 mM tris(2-carboxyethyl)phosphine on the antibody/peptide complex produced in (54-4) 2.0 μL of hydrochloride solution (100 equivalents to antibody) was added and stirred at room temperature for 20 minutes. When the mass was measured by ESI-TOFMS, the starting antibody/peptide complex had heavy chain peaks at 52981 and 53143, and a light chain peak at 23440. A peak was observed at 23440, the same as the starting material, and a light chain peak was observed at 23440.

(summary)
The above results are summarized as follows.

Further, for antibodies, lysine residues at positions 246 and / or 248 (e.g., Examples 8, 15, 44, 46), lysine residues at positions 288 and / or 290 (e.g., Example 42, Examples 48), it was shown that regioselective modification of specific amino acid residues including the lysine residue at position 317 (eg, Example 40) is possible.

Furthermore, the following compounds having an affinity substance for soluble proteins, a cleavable moiety and a reactive group, represented by the following formula (I), will be explained by taking some of the examples as examples, as shown in Table 3 below. That's right.
A-L-B-R (I)
[In the formula,
A is an affinity substance for soluble proteins;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B is (a) a divalent group that includes a bioorthogonal functional group or (b) a divalent group that does not include a bioorthogonal functional group;
R is a reactive group for said soluble protein. ]
L is (i) a cleavable linker that is a divalent group that includes a cleavable moiety that has the ability to generate a bioorthogonal functional group on the side of the reactive group upon cleavage, or (ii) a bioorthogonal functional group upon cleavage is a cleavable linker that is a divalent group containing a cleavable moiety that does not have the ability to generate a on the side of a reactive group.

A variety of compounds having affinity substances for soluble proteins, cleavable moieties and reactive groups, as described above, have an affinity substance for soluble proteins (A), a cleavable linker which is a divalent group comprising a cleavable moiety. Types of (L), (a) divalent groups containing bioorthogonal functional groups, or (b) divalent groups without bioorthogonal functional groups (B), reactive groups for soluble proteins (R) can be prepared according to well-known organic synthesis methods by appropriately changing Further examples of compounds with affinity agents for soluble proteins, cleavable moieties and reactive groups that can be prepared in the present invention and soluble proteins (antibodies) with bioorthogonal functional groups that can be produced thereby The relationship with is as follows.

From the above, in a compound having an affinity substance for a soluble protein, a cleavable moiety and a reactive group, the type of affinity substance, the length of the linker between the affinity substance and the reactive group, and the linker are introduced It was shown that the antibody can be regioselectively modified by appropriately adjusting factors such as the position in the affinity substance.

The present invention is useful, for example, for producing regioselectively modified soluble proteins.

Claims (117)

Formula (I) below:
A-L-B-R (I)
[In the formula,
A is an affinity substance for soluble proteins;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B is (a) a divalent group that includes a bioorthogonal functional group or (b) a divalent group that does not include a bioorthogonal functional group;
R is a reactive group for said soluble protein. ], a compound or a salt thereof having an affinity substance for a soluble protein, a cleavable moiety and a reactive group. L is (i) a cleavable linker that is a divalent group comprising a cleavable moiety capable of generating a bioorthogonal functional group on the side of the reactive group upon cleavage, or (ii) a bioorthogonal functional group upon cleavage. 2. The compound or salt thereof according to claim 1, which is a cleavable linker that is a divalent group comprising a cleavable moiety that does not have the ability to generate on the reactive group side. 3. The compound or salt thereof according to claim 2, wherein L is the cleavable linker of (i). 4. The compound or salt thereof according to claim 2 or 3, wherein L is the cleavable linker of (i) and B is the divalent group of (b). 3. The compound or salt thereof according to claim 2, wherein L is the cleavable linker of (ii) and B is the divalent group of (a). The compound or its salt according to any one of claims 1 to 5, wherein the substance with affinity for soluble proteins is a peptide. 7. The compound or salt thereof according to claim 6, wherein the peptide is a binding peptide to the Fc region of a monoclonal antibody. 8. The compound or salt thereof according to claim 7, wherein the binding peptide is a binding peptide for the Fc region of IgG. Claims 1 to 8, wherein the affinity substance is an affinity substance for an antibody that contains any one Fc region protein selected from the group consisting of the following (A) to (C) and has antigen-binding ability: A compound or salt thereof according to any one of:
(A) an Fc region protein comprising the amino acid sequence of SEQ ID NO: 1;
(B) an Fc region protein comprising an amino acid sequence in which one or several amino acid residues are inserted, added, deleted or substituted in the amino acid sequence of SEQ ID NO: 1; or (C) the amino acid sequence of SEQ ID NO: 1 and 90 An Fc region protein comprising an amino acid sequence exhibiting greater than or equal to % identity. The binding peptide has the following formula (i):
(X _1-3 )-C-(X ₂ )-H-(Xaa1)-G-(Xaa2)-LVWC-(X _1-3 ) (SEQ ID NO: 94) (i)
[In the formula,
X is the same or different and is any amino acid residue other than cysteine;
C is a cysteine residue,
H is a histidine residue,
Xaa1 is an arginine residue, a leucine residue, a lysine residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue;
G is a glycine residue,
Xaa2 is a lysine residue, a glutamine residue, a glutamic acid residue, an asparagine residue, or an aspartic acid residue;
L is a leucine residue,
V is a valine residue and W is a tryptophan residue. ]
A peptide or a salt thereof comprising an amino acid sequence consisting of 13 to 17 amino acid residues and capable of binding to human IgG and / or rabbit IgG, represented by any one of claims 7 to 9. compound or its salt. The binding peptide has the following formula (i-1):
(X _1-3 )-C-(X ₂ )-H-(Xaa1)-G-(Xaa2)-LVWC-(X _1-3 ) (SEQ ID NO: 95) (i-1)
[In the formula,
X is the same or different and is any amino acid residue other than cysteine;
C is a cysteine residue,
H is a histidine residue,
Xaa1 is a lysine residue, a cysteine residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue;
G is a glycine residue,
Xaa2 is a glutamic acid residue or an asparagine residue,
L is a leucine residue,
V is a valine residue and W is a tryptophan residue. ]
A peptide or a salt thereof comprising an amino acid sequence consisting of 13 to 17 amino acid residues and capable of binding to human IgG and / or rabbit IgG, represented by any one of claims 7 to 9. compound or its salt. The binding peptide has the following formula (i-2):
(X _1-3 )-C-(X ₂ )-H-(Xaa1)-G-(Xaa2)-LVWC-(X _1-3 ) (SEQ ID NO: 96) (i-2)
[In the formula,
X is the same or different and is any amino acid residue other than cysteine;
C is a cysteine residue,
H is a histidine residue,
Xaa1 is an arginine residue or a leucine residue,
G is a glycine residue,
Xaa2 is a lysine residue, a glutamine residue, or an aspartic acid residue;
L is a leucine residue,
V is a valine residue, and
W is a tryptophan residue. ] and is capable of binding to human IgG and/or rabbit IgG, or a salt thereof, according to any one of claims 7 to 10. or a salt thereof. The binding peptide has the following formula (v):
(X _1-3 )-C-(Xaa3)-(xaa4)-H-(Xaa1)-G-(Xaa2)-LVWC-(Xaa5)-(Xaa6)-(Xaa7) (sequence number 102)
(v)
[In the formula,
X is the same or different and is any amino acid residue other than cysteine;
C is a cysteine residue,
Xaa3 is an alanine residue or a lysine residue,
Xaa4 is a tryptophan residue or a tyrosine residue,
H is a histidine residue,
Xaa1 is an arginine residue, a leucine residue, a lysine residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue;
G is a glycine residue,
Xaa2 is a lysine residue, a glutamine residue, a glutamic acid residue, an asparagine residue, or an aspartic acid residue;
L is a leucine residue,
V is a valine residue,
W is a tryptophan residue,
Xaa5 is a threonine residue or a lysine residue,
Xaa6 is a tyrosine residue, a lysine residue, or no residue, and Xaa7 is a histidine residue, a lysine residue, or no residue. ] and is capable of binding to human IgG and/or rabbit IgG, or a salt thereof, according to any one of claims 7 to 12. or a salt thereof. The binding peptide has the following formula (vi):
DC-(Xaa3)-(Xaa4)-H-(Xaa1)-G-(Xaa2)-LVWC-(Xaa5)-(Xaa6)-(Xaa7) (SEQ ID NO: 103) (vi ) [In the formula,
D is an aspartic acid residue,
C is a cysteine residue,
Xaa3 is an alanine residue or a lysine residue,
Xaa4 is a tryptophan residue or a tyrosine residue,
H is a histidine residue,
Xaa1 is an arginine residue, a leucine residue, a lysine residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue;
G is a glycine residue,
Xaa2 is a lysine residue, a glutamine residue, a glutamic acid residue, an asparagine residue, or an aspartic acid residue;
L is a leucine residue,
V is a valine residue,
W is a tryptophan residue,
Xaa5 is a threonine residue or a lysine residue,
Xaa6 is a tyrosine residue, a lysine residue, or no residue;
Xaa7 is a histidine residue, a lysine residue, or none. ] and is capable of binding to human IgG and/or rabbit IgG, or a salt thereof, comprising an amino acid sequence consisting of 13 to 15 amino acid residues. A compound according to any one of claims or a salt thereof. The binding peptide has the following formula (vii):
DC-(Xaa3)-(Xaa4)-H-(Xaa1)-G-(Xaa2)-LVWCT (SEQ ID NO: 104) (vii)
[In the formula,
D is an aspartic acid residue,
C is a cysteine residue,
Xaa3 is an alanine residue or a lysine residue,
Xaa4 is a tryptophan residue or a tyrosine residue,
H is a histidine residue,
Xaa1 is an arginine residue, a leucine residue, a lysine residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue;
G is a glycine residue,
Xaa2 is a lysine residue, a glutamine residue, a glutamic acid residue, an asparagine residue, or an aspartic acid residue;
L is a leucine residue,
V is a valine residue,
W is a tryptophan residue, and T is a threonine residue], and is capable of binding to human IgG and/or rabbit IgG. The compound or its salt according to any one of claims 7 to 14, which is a characterized peptide or its salt. The binding peptide has the following formula (viii):
RGNC-(Xaa3)-(Xaa4)-H-(Xaa1)-G-(Xaa2)-LVWC-(Xaa5)-(Xaa6)-(Xaa7) (SEQ ID NO: 105) (viii)
[In the formula,
R is an arginine residue,
G is a glycine residue,
N is an asparagine residue,
C is a cysteine residue,
Xaa3 is an alanine residue or a lysine residue,
Xaa4 is a tryptophan residue or a tyrosine residue,
H is a histidine residue,
Xaa1 is an arginine residue, a leucine residue, a lysine residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue;
G is a glycine residue,
Xaa2 is a lysine residue, a glutamine residue, a glutamic acid residue, an asparagine residue, or an aspartic acid residue;
L is a leucine residue,
V is a valine residue,
W is a tryptophan residue,
Xaa5 is a threonine residue or a lysine residue,
Xaa6 is a tyrosine residue, a lysine residue, or no residue, and Xaa7 is a histidine residue, a lysine residue, or no residue. ] and is capable of binding to human IgG and/or rabbit IgG, or a salt thereof, comprising an amino acid sequence consisting of 13 to 15 amino acid residues. A compound according to any one of claims or a salt thereof. The compound or its salt according to any one of claims 7 to 16, wherein the binding peptide is capable of binding to human IgG. In the amino acid sequence of (a) FNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 92), any of the amino acid residues of the binding peptide is a lysine residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, and a diamino acid residue. Affinity comprising an amino acid sequence substituted by one amino acid residue selected from the group consisting of propionic acid residues and (b) having 90% or more identity to said amino acid sequence of SEQ ID NO:92 The compound or its salt according to any one of claims 7 to 9, which is a peptide or its salt. The cleavable moiety is (a) treated with one or more substances selected from the group consisting of acidic substances, basic substances, reducing agents, oxidizing agents, and enzymes, and (b) physicochemical stimulation selected from the group consisting of light. or (c) left alone when using a cleavable linker comprising a self-cleavable cleavable moiety, or the salt. The cleavable moiety is a disulfide residue, an acetal residue, a ketal residue, an ester residue, a carbamoyl residue, an alkoxyalkyl residue, an imine residue, a tertiary alkyloxycarbamate residue, a silane residue, a hydrazone-containing residue. groups, phosphoramidate residues, aconityl residues, trityl residues, azo residues, vicinal diol residues, selenium residues, aromatic ring-containing residues with electron-withdrawing groups, coumarin-containing residues, sulfone-containing 20. The compound or salt thereof according to any one of claims 1 to 19, which is selected from the group consisting of residues, unsaturated bond-containing chain residues, and glycosyl residues. 21. Any one of claims 2 to 20, wherein the cleavable moiety of (i) is selected from the group consisting of disulfide residues, ester residues, acetal residues, ketal residues, imine residues and vicinal diol residues. A compound or a salt thereof according to claim 1. The cleavable moiety of (ii) is an ester residue, a carbamoyl residue, an alkoxyalkyl residue, an imine residue, a tertiary alkyloxycarbamate residue, a silane residue, a hydrazone-containing residue, a phosphoramidate residue. , aconityl residue, trityl residue, azo residue, vicinal diol residue, selenium residue, aromatic ring-containing residue with electron-withdrawing group, coumarin-containing residue, sulfone-containing residue, unsaturated bond-containing chain The compound or salt thereof according to any one of claims 2 to 20, which is selected from the group consisting of residues and glycosyl residues. The cleavable portion is:

[Where the wavy line perpendicular to the bond indicates the cleavage site,
multiple R _2a , multiple R _2b , and multiple R _2c are the same or different,
(i) a hydrogen atom, or a halogen atom;
(ii) a monovalent hydrocarbon group;
(iii) aralkyl;
(iv) a monovalent heterocyclic group;
(v) R _c -O-, R _c -C(=O)-, R _c -O-C(=O)-, or R _c -C(=O)-O- (R _c is a hydrogen atom , or a monovalent hydrocarbon group);
(vi) NR _d R _e -, NR _d R _e -C(=O)-, NR _d R _e -C(=O)-O-, or R _d -C(=O)-NR _e -(R _d and R _e are the same or different and represent a hydrogen atom or a monovalent hydrocarbon group); or (vii) from the group consisting of a nitro group, a sulfate group, a sulfonate group, a cyano group, or a carboxyl group chosen,
J is —CH ₂ —, —O—, or —S—;
r is any integer from 1 to 4,
O (white circle) indicates binding to A, and ● (filled circle) indicates binding to B.
If the chemical structure is asymmetric about the cleavage site, the circle may indicate the bond to A and the circle may indicate the bond to B. 21. The compound or salt thereof according to any one of claims 1 to 20, which corresponds to any one chemical structure selected from the group consisting of: The cleavable moiety of (i) above is:

[Where the wavy line perpendicular to the bond indicates the cleavage site,
R _2a is the same as in claim 23,
O (white circle) indicates binding to A, and ● (filled circle) indicates binding to B.
If the chemical structure is asymmetric about the cleavage site, the circle may indicate the bond to A and the circle may indicate the bond to B. The compound or salt thereof according to any one of claims 2 to 19, 21 and 23, which corresponds to any one chemical structure selected from the group consisting of: The cleavable moiety of (ii) above is:

[Where the wavy line perpendicular to the bond indicates the cleavage site,
R _2b , R _2c , J, r are the same as in claim 23,
O (white circle) indicates binding to A, and ● (filled circle) indicates binding to B.
If the chemical structure is asymmetric about the cleavage site, the circle may indicate the bond to A and the circle may indicate the bond to B. 24. The compound or salt thereof according to any one of claims 2 to 19, 22 and 23, which corresponds to any one chemical structure selected from the group consisting of: L is the following formulas (L1) to (L3):
La-C-Lb (L1)
La-C (L2)
C-Lb (L3)
[In the formula,
La and Lb are each a divalent group,
C is a cleavable moiety. ], the compound or a salt thereof according to any one of claims 1 to 25. Said La and Lb are respectively the following (La') and (Lb'):

[In the formula,
p and p' are the same or different and any integer from 0 to 10;
q and q' are the same or different and any integer from 0 to 10;
X and X' are the same or different and are a carbon atom, a nitrogen atom, or a single bond (wherein when X is a nitrogen atom, R _1b is absent, and when X' is a nitrogen atom, R _{1b '} is absent.If X is a single bond, R _1a and R _1b are absent, and if X' is a single bond, R _1a' and R _1b' are absent);
R _1a , R _1b , R _1a′ and R _1b′ are the same or different and are atoms or groups selected from the group consisting of (i) to (vii). ], The compound or its salt of Claim 26. A bivalent group containing a bioorthogonal functional group is an azide residue, an aldehyde residue, a thiol residue, an alkyne residue, an alkene residue, a tetrazine residue, a nitrone residue, a hydroxylamine residue, a nitrile residue, hydrazine residue, ketone residue, boronic acid residue, cyanobenzothiazole residue, allyl residue, phosphine residue, maleimide residue, disulfide residue, thioester group, α-halocarbonyl residue, isonitrile residue, sydone 28. The compound or salt thereof according to any one of claims 1 to 27, which is a divalent group containing a bioorthogonal functional group selected from the group consisting of residues and selenium residues in its main chain. A bivalent group containing a bioorthogonal functional group is an azide residue, an aldehyde residue, a thiol residue, an alkyne residue, an alkene residue, a halogen residue, a tetrazine residue, a nitrone residue, a hydroxylamine residue, nitrile residue, hydrazine residue, ketone residue, boronic acid residue, cyanobenzothiazole residue, allyl residue, phosphine residue, maleimide residue, disulfide residue, α-halocarbonyl residue, isonitrile residue, 28. The compound or salt thereof according to any one of claims 1 to 27, which is a divalent group containing a bioorthogonal functional group selected from the group consisting of a sydnon residue and a selenium residue in its side chain. The bioorthogonal functional groups are:

[In the formula,
R _1f , single or multiple R _1g and single or multiple R _1h are the same or different and are atoms or groups selected from the group consisting of (i) to (vii), or electron-withdrawing groups;
・ is a bond. ), the compound or a salt thereof according to any one of claims 1 to 29. The divalent group (b) is optionally substituted alkylene, optionally substituted cycloalkylene, optionally substituted aryl, optionally substituted divalent heterocyclic group, - NR _a — (R _a represents a hydrogen atom or a substituent), —O—, or the compound according to any one of claims 1 to 30, or a compound thereof selected from the group consisting of a combination of two or more thereof salt. B is the following formula (B-1):

[In the formula,
Y is -NH-, -O-, -CH ₂ -, or the following formula (B-2):

(In the formula,
V and V′ are the same or different and are —NH—, —O—, —CH ₂ —, or a single bond;
V is a divalent group containing a bioorthogonal functional group;
s is any integer from 0 to 10,
○ and ● in formula (B-2) have the same orientations as ○ and ● in formula (B-1), respectively. ) and
Z is an oxygen atom, a sulfur atom, or a hydrogen atom (when Z is a hydrogen atom, -C(=Z)- represents -CH ₂ -);
○ (white circle) in formula (B-1) indicates a bond to the L-side portion, and ● (black circle) indicates a bond to the R-side portion. ], the compound or a salt thereof according to any one of claims 1 to 31. 33. The compound of any one of claims 1-32, wherein the reactive group is a reactive group specific for the side chain of any one of a lysine residue, a tyrosine residue, or a tryptophan residue, or the salt. 34. The compound or salt thereof according to claim 33, wherein the reactive group is a reactive group specific to the side chain of a lysine residue. The reactive groups are:

〔here,
R _5a and R _5c are atoms or groups selected from the group consisting of (i) to (vii);
R _5b is an electron withdrawing group,
j is any integer from 1 to 5;
k is any integer from 1 to 4. 35. The compound or salt thereof according to any one of claims 1 to 34, which corresponds to any one chemical structure selected from the group consisting of: 36. The compound or salt thereof according to any one of claims 1 to 35, wherein the main chain connecting A and R has 4 to 20 atoms. The compound or salt thereof according to any one of claims 1 to 36, wherein the main chain connecting A and R does not contain a ring structure. 38. The compound or salt thereof according to any one of claims 1 to 37, wherein the partial structure represented by LB does not contain a peptide moiety. The compound represented by the formula (I) is the following (I'):
AB2-L'-B1-R (I')
[In the formula,
A and R are the same as in formula (I) above L′ is a cleavable linker that is a divalent group containing a cleavable moiety;
B1 and B2 are the same or different and are (a) a divalent group containing a bioorthogonal functional group or (b) a divalent group containing no bioorthogonal functional group;
B1 and B2 may have a symmetrical structure about L'. ], the compound or a salt thereof according to any one of claims 1 to 38. The compound represented by the formula (I′) is the following (I″):

[In the formula,
A and R are the same as those of formula (I) described in claim 1,
C is a cleavable moiety;
p, p', q, q', X, X', R _1a , R _1a' , R _1b and R _1b' are the same as in formulas (La') and (Lb') according to claim 27 and
Y and Y' are the same or different and are the same as Y in formula (B-1) according to claim 32,
Z and Z' are the same or different and are the same as Z in formula (B-1) above. 40. A compound or a salt thereof according to any one of claims 39, which is represented by: Formula (I) below:
A-L-B-R (I)
[In the formula,
A is an affinity substance for soluble proteins;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B is (a) a divalent group that includes a bioorthogonal functional group or (b) a divalent group that does not include a bioorthogonal functional group;
R is a reactive group for said soluble protein. ], a regioselective modification reagent for a soluble protein comprising a compound having an affinity for a soluble protein, a cleavable moiety and a reactive group or a salt thereof. Formula (I) below:
A-L-B-R (I)
[In the formula,
A is an affinity substance for the antibody;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B is (a) a divalent group that includes a bioorthogonal functional group or (b) a divalent group that does not include a bioorthogonal functional group;
R is a reactive group specific for the side chains of lysine residues. ], a compound or a salt thereof having an affinity substance for an antibody, a cleavable moiety and a reactive group. Formula (I) below:
A-L-B-R (I)
[In the formula,
A is an affinity substance for the antibody;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B is (a) a divalent group that includes a bioorthogonal functional group or (b) a divalent group that does not include a bioorthogonal functional group;
R is a reactive group specific for the side chains of lysine residues. ], an antibody regioselective modification reagent comprising an antibody-affinity substance, a cleavable moiety and a compound having a reactive group or a salt thereof. Formula (II) below:
A-LB-R'-T (II)
[In the formula,
A is an affinity substance for soluble proteins;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B is (a) a divalent group that includes a bioorthogonal functional group or (b) a divalent group that does not include a bioorthogonal functional group;
R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein. ] and a soluble protein having a cleavable moiety or a salt thereof. 45. The soluble protein or salt thereof of claim 44, wherein the soluble protein is a monoclonal antibody. 46. The soluble protein or salt thereof according to claim 44 or 45, wherein the soluble protein is an IgG antibody. The soluble protein or salt thereof according to any one of claims 44 to 46, wherein the soluble protein is of human origin. The soluble protein comprises any one Fc region protein selected from the group consisting of the following (A) to (C) and is an antibody having antigen-binding ability, according to any one of claims 44 to 47 Soluble protein or its salt:
(A) an Fc region protein comprising the amino acid sequence of SEQ ID NO: 1;
(B) an Fc region protein comprising an amino acid sequence in which one or several amino acid residues are inserted, added, deleted or substituted in the amino acid sequence of SEQ ID NO: 1; or (C) the amino acid sequence of SEQ ID NO: 1 and 90 An Fc region protein comprising an amino acid sequence exhibiting greater than or equal to % identity. The soluble protein contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and the specific amino acid residue in a non-target region other than the target region including 5 or more
The structural unit represented by ALBR' binds to one or more specific amino acid residues contained in the target region with a regioselectivity of 30% or more. The soluble protein or salt thereof according to any one of 44-48. The soluble protein or salt thereof according to claim 49, wherein said target region is a region consisting of 1-10 contiguous amino acid residues. 51. The soluble protein or salt thereof according to claim 50, wherein said target region is a region consisting of 1-3 contiguous amino acid residues. The target region is (a) a region consisting of amino acid residues at positions 246-248 in the human IgG Fc region, (b) a region consisting of amino acid residues at positions 288-290 in the human IgG Fc region, or (c) human 52. The soluble protein or salt thereof according to claim 51, which is a region consisting of amino acid residue 317 in the IgG Fc region. The soluble protein or salt thereof according to any one of claims 49 to 52, wherein said regioselectivity is 50% or more. 54. The soluble protein or salt thereof according to claim 53, wherein said regioselectivity is 70% or more. 55. The soluble protein or salt thereof according to claim 54, wherein said regioselectivity is 90% or more. The specific amino acid residue is a (where a is an arbitrary integer of 1 to 10) on each of the N-terminal side and the C-terminal side centering on a specific amino acid present at a specific position. Any of claims 49 to 55, which does not contain amino acid residues of the same type as the specific amino acid residue other than the specific amino acid residue present at the specific position in the region to the distant position of the amino acid residue of A soluble protein or a salt thereof according to claim 1. the soluble protein is a multimeric protein comprising a plurality of monomeric proteins;
T has a structural unit represented by ALBR' in a plurality of corresponding target regions in a plurality of monomeric proteins, so that the multimeric protein is ALB- The soluble protein or salt thereof according to any one of claims 44 to 56, which has a plurality of structural units represented by R'. wherein said soluble protein is an antibody comprising multiple heavy chains;
T has a structural unit represented by ALBR' in a plurality of corresponding target regions in a plurality of heavy chains, such that the antibody is represented by ALBR'. The soluble protein or salt thereof according to any one of claims 44 to 57, having a plurality of structural units. 59. The soluble protein or salt thereof of claim 58, wherein the number of heavy chains is two. a side chain of any one of a lysine residue, a tyrosine residue, or a tryptophan residue for a lysine residue, a tyrosine residue, or a tryptophan residue, where the moiety generated by the reaction between the soluble protein and the reactive group is a lysine, tyrosine, or tryptophan residue; The soluble protein or salt thereof according to any one of claims 44 to 59, which is a moiety generated by reaction of a reactive group specific for wherein the moiety generated by reaction between a soluble protein and a reactive group is a moiety generated by reaction between a lysine residue and a reactive group specific for the side chain of the lysine residue. The soluble protein or salt thereof according to any one of Items 44-60. The moieties produced by the reaction are:

[Here, ● (black circle) indicates a bond to the portion on the T side, and O (white circle) indicates a bond to the portion on the B side. A straight line perpendicular to the bond indicates the bond produced by the reaction. 62. The soluble protein or salt thereof according to any one of claims 44 to 61, which corresponds to any one chemical structure selected from the group consisting of: The soluble protein or salt thereof according to any one of claims 44 to 62, wherein the main chain connecting A and R' has 4 to 20 atoms. The soluble protein or salt thereof according to any one of claims 44 to 63, wherein the main chain connecting A and R does not contain a ring structure. The soluble protein or salt thereof according to any one of claims 44 to 64, wherein the partial structure represented by LB does not contain a peptide portion. The compound represented by the formula (II) is the following (II'):
AB2-L'-B1-R'-T (II')
[In the formula,
A, R′ and T are the same as in formula (II) above L′ is a cleavable linker that is a divalent group containing a cleavable moiety;
B1 and B2 are the same or different and are (a) a divalent group containing a bioorthogonal functional group or (b) a divalent group containing no bioorthogonal functional group;
B1 and B2 may have a symmetrical structure about L'. ], the soluble protein or a salt thereof according to any one of claims 44 to 65. The compound represented by the formula (II') is the following (II''):

[In the formula,
A, R' and T are the same as in formula (II) according to claim 44;
C is a cleavable moiety;
p and p' are the same or different and any integer from 0 to 10;
q and q' are the same or different and any integer from 0 to 10;
X and X' are the same or different and are a carbon atom, a nitrogen atom, or a single bond (wherein when X is a nitrogen atom, R _1b is absent, and when X' is a nitrogen atom, R _{1b '} is absent.If X is a single bond, R _1a and R _1b are absent, and if X' is a single bond, R _1a' and R _1b' are absent);
R _1a , R _1b , R _1a′ and R _1b′ are the same or different,
(i) a hydrogen atom, or a halogen atom;
(ii) a monovalent hydrocarbon group;
(iii) aralkyl;
(iv) a monovalent heterocyclic group;
(v) R _c -O-, R _c -C(=O)-, R _c -O-C(=O)-, or R _c -C(=O)-O- (R _c is a hydrogen atom , or a monovalent hydrocarbon group);
(vi) NR _d R _e -, NR _d R _e -C(=O)-, NR _d R _e -C(=O)-O-, or R _d -C(=O)-NR _e -(R _d and R _e are the same or different and represent a hydrogen atom or a monovalent hydrocarbon group); or (vii) from the group consisting of a nitro group, a sulfate group, a sulfonate group, a cyano group, or a carboxyl group chosen,
Y and Y' are the same or different and are the same as Y in formula (B-1) according to claim 32,
Z and Z' are the same or different and are the same as Z in the formula (B-1) described in claim 32. ], the soluble protein or its salt according to claim 66. Formula (II) below:
A-LB-R'-T (II)
[In the formula,
A is an affinity substance for the antibody;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B is (a) a divalent group that includes a bioorthogonal functional group or (b) a divalent group that does not include a bioorthogonal functional group;
R' is a moiety generated by reaction between an antibody and a reactive group specific for the side chain of a lysine residue;
T is an antibody. ] and an antibody or salt thereof having a cleavable moiety, represented by an affinity substance for an antibody. A method for producing a soluble protein affinity substance and a soluble protein or salt thereof having a cleavable moiety, comprising:
Formula (I) below:
A-L-B-R (I)
[In the formula,
A is an affinity substance for soluble proteins;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B is (a) a divalent group that includes a bioorthogonal functional group or (b) a divalent group that does not include a bioorthogonal functional group;
R is a reactive group for said soluble protein. ], a compound having an affinity for soluble proteins, a cleavable moiety and a reactive group or a salt thereof, is reacted with a soluble protein,
Formula (II) below:
A-LB-R'-T (II)
[In the formula,
A, L, and B are the same as in formula (I) above;
R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein. and a soluble protein or salt thereof having a cleavable moiety. 70. The method of claim 69, wherein the soluble protein is an antibody and the reactive group is a reactive group specific for side chains of lysine residues. Formula (III) below:
ALB'(-F)-R'-T (III)
[In the formula,
A is an affinity substance for soluble proteins;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B' is a divalent group that includes a moiety generated by a reaction between a functional agent and a bioorthogonal functional group;
F is a functional substance,
R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein. ], a complex having an affinity substance for a soluble protein, a cleavable moiety, a functional substance and a soluble protein, or a salt thereof. 72. The conjugate or salt thereof of claim 71, wherein the soluble protein is a monoclonal antibody. 73. The conjugate or salt thereof according to claim 71 or 72, wherein the soluble protein is an IgG antibody. The complex or salt thereof according to any one of claims 71 to 73, wherein the soluble protein is of human origin. The soluble protein comprises any one Fc region protein selected from the group consisting of the following (A) to (C) and is an antibody having antigen-binding ability, according to any one of claims 71 to 74 Complexes or their salts:
(A) an Fc region protein comprising the amino acid sequence of SEQ ID NO: 1;
(B) an Fc region protein comprising an amino acid sequence in which one or several amino acid residues are inserted, added, deleted or substituted in the amino acid sequence of SEQ ID NO: 1; or (C) the amino acid sequence of SEQ ID NO: 1 and 90 An Fc region protein comprising an amino acid sequence exhibiting greater than or equal to % identity. The soluble protein contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and the specific amino acid residue in a non-target region other than the target region including 5 or more
The structural unit represented by ALB'(-F)-R' binds to one or more specific amino acid residues contained in the target region with a position selectivity of 30% or more. The conjugate or salt thereof according to any one of claims 71 to 75, wherein The specific amino acid residue is a (where a is an arbitrary integer of 1 to 10) on each of the N-terminal side and the C-terminal side centering on a specific amino acid present at a specific position. 77. The complex or the salt. the soluble protein is a multimeric protein comprising a plurality of monomeric proteins;
T has a structural unit represented by ALB'(-F)-R' in a plurality of corresponding target regions in a plurality of monomeric proteins. As a result, the multimeric protein is A 78. The complex or salt thereof according to any one of claims 71 to 77, which has a plurality of structural units represented by -LB'(-F)-R'. wherein said soluble protein is an antibody comprising multiple heavy chains;
T has a structural unit represented by ALB'(-F)-R' in a plurality of corresponding target regions in a plurality of heavy chains, such that the antibody has ALB The complex or salt thereof according to any one of claims 71 to 78, which has a plurality of structural units represented by '(-F)-R'. The divalent group containing the portion generated by the reaction between the functional substance and the bioorthogonal functional group is a disulfide residue, an acetal residue, a ketal residue, an ester residue, a carbamoyl residue, an alkoxyalkyl residue. , imine residues, tertiary alkyloxycarbamate residues, silane residues, hydrazone-containing residues, phosphoramidate residues, aconityl residues, trityl residues, azo residues, vicinal diol residues, selenium residues , an aromatic ring-containing residue having an electron-withdrawing group, a coumarin-containing residue, a sulfone-containing residue, an unsaturated bond-containing chain residue, and a glycosyl residue. , the complex or salt thereof according to any one of claims 71 to 79. The moieties produced by the reaction are:

[Here, the wavy line orthogonal to the bond indicates the bond generated by the reaction,
multiple R _2a , multiple R _2b , and multiple R _2c are the same or different,
(i) a hydrogen atom, or a halogen atom;
(ii) a monovalent hydrocarbon group;
(iii) aralkyl;
(iv) a monovalent heterocyclic group;
(v) R _c -O-, R _c -C(=O)-, R _c -O-C(=O)-, or R _c -C(
=O)-O- (R _c represents a hydrogen atom or a monovalent hydrocarbon group);
(vi) NR _d R _e -, NR _d R _e -C(=O)-, NR _d R _e -C(=O)-O-, or R _d -C(=O)-NR _e -(R _d and R _e are the same or different and represent a hydrogen atom or a monovalent hydrocarbon group); or (vii) from the group consisting of a nitro group, a sulfate group, a sulfonate group, a cyano group, or a carboxyl group chosen,
J is —CH ₂ —, —O—, or —S—;
r is any integer from 1 to 4,
O (white circle) indicates binding to A, and ● (filled circle) indicates binding to B.
If the chemical structure is asymmetric about the cleavage site, the circle may indicate the bond to A and the circle may indicate the bond to B. 81. The complex or salt thereof according to any one of claims 71 to 80, which corresponds to any one chemical structure selected from the group consisting of: The portion generated by the reaction between the soluble protein and the reactive group is a lysine residue, a tyrosine residue, or a tryptophan residue and a side chain of any one of a lysine residue, a tyrosine residue, or a tryptophan residue. 82. The conjugate or salt thereof according to any one of claims 71 to 81, which is a moiety generated by reaction between a reactive group specific for wherein the moiety generated by reaction between a soluble protein and a reactive group is a moiety generated by reaction between a lysine residue and a reactive group specific for the side chain of the lysine residue. The complex or salt thereof according to any one of Items 71 to 82. The moieties produced by the reaction are:

[Here, ● (black circle) indicates a bond to the portion on the T side, and O (white circle) indicates a bond to the portion on the B side. 84. The complex or salt thereof according to any one of claims 71 to 83, which corresponds to any one chemical structure selected from the group consisting of: The complex or salt thereof according to any one of claims 71 to 84, wherein the functional substance is a drug or labeling substance. The complex or salt thereof according to any one of claims 71 to 85, wherein the functional substance is a low-molecular-weight compound. 87. The conjugate or salt thereof according to claim 85 or 86, wherein the drug is an anticancer agent. The complex or salt thereof according to any one of claims 71 to 87, wherein the main chain connecting A and R' has 4 to 20 atoms. The complex or salt thereof according to any one of claims 71 to 88, wherein the main chain connecting A and R does not contain a ring structure. The complex or salt thereof according to any one of claims 71 to 89, wherein the partial structure represented by LB does not contain a peptide moiety. The compound represented by the formula (III) is represented by the following formula (III'):
A-B2'(-F2)-L'-B1'(-F1)-R'-T (III')
[In the formula,
A, R' and T are the same as in formula (II) above;
L' is a cleavable linker that is a divalent group containing a cleavable moiety;
B1' and B2' are the same or different and are divalent groups comprising moieties generated by reaction between a functional agent and a bioorthogonal functional group;
F1 and F2 are the same or different and are functional substances;
B1' (-F1) and B2' (-F2) may have a symmetrical structure about L'. ], the complex or a salt thereof according to any one of claims 71 to 90. The compound represented by the formula (III') is the following (III''):

[In the formula,
A, R' and T are the same as in formula (III) according to claim 71;
C is a cleavable moiety;
p and p' are the same or different and any integer from 0 to 10;
q and q' are the same or different and any integer from 0 to 10;
X and X' are the same or different and are a carbon atom, a nitrogen atom, or a single bond (wherein when X is a nitrogen atom, R _1b is absent, and when X' is a nitrogen atom, R _{1b '} is absent.If X is a single bond, R _1a and R _1b are absent, and if X' is a single bond, R _1a' and R _1b' are absent);
R _1a , R _1b , R _1a′ and R _1b′ are the same or different and are selected from the group consisting of (i) to (vii);
Y and Y' are the same or different and are a residue obtained by removing one hydrogen atom from Y in formula (B-1) according to claim 32,
Z and Z' are the same or different and are the same as Z in the formula (B-1);
F and F' are the same or different and are functional substances. ], or a salt thereof according to claim 91. Formula (III) below:
ALB'(-F)-R'-T (III)
[In the formula,
A is an affinity substance for the antibody;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B' is a divalent group that includes a moiety generated by a reaction between a functional agent and a bioorthogonal functional group;
F is a functional substance,
R' is a moiety generated by reaction between an antibody and a reactive group specific for the side chain of a lysine residue;
T is an antibody. ], a complex having an affinity substance, a functional substance and an antibody or a salt thereof. A method for producing a complex or a salt thereof having an affinity substance for a soluble protein, a cleavable moiety, a functional substance and a soluble protein,
Formula (II) below:
A-LB-R'-T (II)
[In the formula,
A is an affinity substance for soluble proteins;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B is (a) a divalent group comprising a bioorthogonal functional group;
R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein. ] and a soluble protein having a cleavable moiety or a salt thereof with a functional substance,
Formula (III) below:
ALB'(-F)-R'-T (III)
[In the formula,
A, L, R' and T are the same as in formula (II) above;
B' is a divalent group that includes a moiety generated by a reaction between a functional agent and a bioorthogonal functional group;
F is a functional substance. ], a conjugate having an affinity agent for a soluble protein, a cleavable moiety, a functional agent and a soluble protein, or a salt thereof. 95. The method of claim 94, wherein the soluble protein is an antibody and the reactive group is a reactive group specific for side chains of lysine residues. A method for producing a complex or a salt thereof having an affinity substance for a soluble protein, a cleavable moiety, a functional substance and a soluble protein,
(A) the following formula (I):
A-L-B-R (I)
[In the formula,
A is an affinity substance for soluble proteins;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B is (a) a divalent group comprising a bioorthogonal functional group;
R is a reactive group for said soluble protein. ], the compound or its salt is reacted with a soluble protein,
Formula (II) below:
A-LB-R'-T (II)
[In the formula,
A, L, and B are the same as in formula (I) above R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein. and (B) an affinity substance for said soluble protein and a soluble protein having a cleavable moiety or reacting the salt with a functional substance,
Formula (III) below:
ALB'(-F)-R'-T (III)
[In the formula,
A and L are the same as in formula (I) above;
R' and T are the same as in formula (II) above;
B' is a divalent group that includes a moiety generated by a reaction between a functional agent and a bioorthogonal functional group;
F is a functional substance. ], a conjugate having an affinity agent for a soluble protein, a cleavable moiety, a functional agent and a soluble protein, or a salt thereof. A method for producing a soluble protein or a salt thereof having a bioorthogonal functional group, comprising:
Formula (II) below:
A-LB-R'-T (II)
[In the formula,
A is an affinity substance for soluble proteins;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B is (a) a divalent group that includes a bioorthogonal functional group or (a) a divalent group that does not include a bioorthogonal functional group;
R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein. ], cleaving the cleavable portion of a soluble protein having an affinity for soluble proteins and a soluble protein having a cleavable portion or a salt thereof,
Formula (IV) below:
L1-B-R'-T (IV)
[In the formula,
B, R' and T are the same as in formula (II) above;
L1 is (i') a monovalent group that contains a bioorthogonal functional group or (ii') a monovalent group that does not contain a bioorthogonal functional group. ] or a salt thereof having a bioorthogonal functional group. L is (i) a cleavable linker that is a divalent group comprising a cleavable moiety capable of generating a bioorthogonal functional group on the side of the reactive group upon cleavage, or (ii) a bioorthogonal functional group upon cleavage. 97. The method of claim 96, wherein the cleavable linker is a divalent group comprising a cleavable moiety that does not have the ability to generate on the side of a reactive group. L is the cleavable linker of (i) above,
L1 is (i′) a monovalent group containing a bioorthogonal functional group;
99. The method of claim 98, wherein B is the divalent group of (a) or (b). L is the cleavable linker of (i) above,
L1 is (i′) a monovalent group containing a bioorthogonal functional group;
100. The method of claim 98 or 99, wherein B is the divalent group of (b). L is the cleavable linker of (ii) above,
L1 is (i′) a monovalent group that does not contain a bioorthogonal functional group;
99. The method of claim 98, wherein B is the divalent group of (a). 102. The method of any one of claims 97-101, wherein the soluble protein is an antibody and the reactive group is a reactive group specific for the side chains of lysine residues. A method for producing a soluble protein or a salt thereof having a bioorthogonal functional group, comprising:
(A) the following formula (I):
A-L-B-R (I)
[In the formula,
A is an affinity substance for soluble proteins;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B is (a) a divalent group that includes a bioorthogonal functional group or (a) a divalent group that does not include a bioorthogonal functional group;
R is a reactive group for said soluble protein. ], the compound or its salt is reacted with a soluble protein,
Formula (II) below:
A-LB-R'-T (II)
[In the formula,
A, L, and B are the same as in formula (I) above R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein. and (B) an affinity substance for said soluble protein and a soluble protein having a cleavable moiety or cutting the cleavable portion of the salt,
Formula (IV) below:
L1-B-R'-T (IV)
[In the formula,
B, R' and T are the same as in formula (II) above;
L1 is (i') a monovalent group that contains a bioorthogonal functional group or (ii') a monovalent group that does not contain a bioorthogonal functional group. ] or a salt thereof having a bioorthogonal functional group. A method for producing a soluble protein having a functional substance or a salt thereof,
Formula (III) below:
ALB'(-F)-R'-T (III)
[In the formula,
A is an affinity substance for soluble proteins;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B' is a divalent group that includes a moiety generated by a reaction between a functional agent and a bioorthogonal functional group;
F is a functional substance,
R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein. ], or cleaving the cleavable portion of a complex having an affinity substance for a soluble protein, a cleavable portion, a functional substance and a soluble protein or a salt thereof, or the following formula (IV):
L1-B-R'-T (IV)
[In the formula,
L1 is (i′) a monovalent group containing a bioorthogonal functional group or (ii′) a monovalent group not containing a bioorthogonal functional group;
B is (a) a divalent group that includes a bioorthogonal functional group or (b) a divalent group that does not include a bioorthogonal functional group;
R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein. ], by reacting a soluble protein having a bioorthogonal functional group or a salt thereof with a functional substance,
Formula (V) below:
F-(L1-B)'-R'-T (V)
[In the formula,
L1 is (i′) a monovalent group containing a bioorthogonal functional group or (ii′) a monovalent group not containing a bioorthogonal functional group;
B is (a) a divalent group that includes a bioorthogonal functional group or (b) a divalent group that does not include a bioorthogonal functional group;
The structural unit represented by (L1-B)' includes a moiety generated by a reaction between a functional substance and either one or both of the bioorthogonal functional groups in (i') and (a). is a divalent structural unit,
F is a functional substance,
R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein. ], comprising producing a soluble protein having a functional substance or a salt thereof. cleaving the cleavable portion of a complex having an affinity substance for a soluble protein, a cleavable portion, a functional substance and a soluble protein or a salt thereof,
The following formula (V1):
L1-B'(-F)-R'-T (V1)
[In the formula,
L1 is (i′) a monovalent group containing a bioorthogonal functional group or (ii′) a monovalent group not containing a bioorthogonal functional group;
B', F, R' and T are the same as in formula (III) above. 105. The method according to claim 104, which is a method comprising producing a soluble protein having a functional substance represented by ] or a salt thereof. reacting the soluble protein having a bioorthogonal functional group or a salt thereof with one or two functional substances,
The following formula (V2):
FL1'-B-R'-T (V2)
[In the formula,
B, R' and T are the same as in formula (IV) above;
L1' is a divalent group comprising a moiety produced by a reaction between a functional agent and (i') a monovalent group comprising a bioorthogonal functional group;
F is a functional substance. ], or the following formula (V3):
Fa-L1'-B'(-Fb)-R'-T (V3)
[In the formula,
R' and T are the same as in formula (IV) above;
L1' is the same as in formula (V2) above,
B' is a divalent group that includes a moiety generated by a reaction between a functional agent and a bioorthogonal functional group;
Fa and Fb are the same or different functional substances, respectively. 105. The method according to claim 104, which is a method comprising producing a soluble protein having a functional substance represented by ] or a salt thereof. A method according to any one of claims 104 to 106, wherein the soluble protein is an antibody and the reactive group is a reactive group specific for side chains of lysine residues. A method for producing a soluble protein having a functional substance or a salt thereof,
(A) the following formula (II):
A-LB-R'-T (II)
[In the formula,
A is an affinity substance for soluble proteins;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B is (a) a divalent group comprising a bioorthogonal functional group;
R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein. ] and a soluble protein having a cleavable moiety or a salt thereof with a functional substance,
Formula (III) below:
ALB'(-F)-R'-T (III)
[In the formula,
A, L, R' and T are the same as in formula (II) above;
B' is a divalent group that includes a moiety generated by a reaction between a functional agent and a bioorthogonal functional group;
F is a functional substance. ] and (B) the affinity substance for the soluble protein, the cleavable moiety , cleaving a cleavable portion of a complex having a functional agent and a soluble protein or a salt thereof,
The following formula (V1):
L1-B'(-F)-R'-T (V1)
[In the formula,
L1 is (i′) a monovalent group containing a bioorthogonal functional group or (ii′) a monovalent group not containing a bioorthogonal functional group;
B', F, R' and T are the same as in formula (III) above. ], comprising producing a soluble protein having a functional substance or a salt thereof. A method for producing a soluble protein having a functional substance or a salt thereof,
(A) the following formula (I):
A-L-B-R (I)
[In the formula,
A is an affinity substance for soluble proteins;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B is (a) a divalent group comprising a bioorthogonal functional group;
R is a reactive group for said soluble protein. ], a compound having an affinity for soluble proteins, a cleavable moiety and a reactive group or a salt thereof, is reacted with a soluble protein,
Formula (II) below:
A-LB-R'-T (II)
[In the formula,
A, L, and B are the same as in formula (I) above R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein. ], and producing a soluble protein or a salt thereof having a cleavable moiety,
(B) reacting an affinity substance for the soluble protein and a soluble protein having a cleavable moiety or a salt thereof with a functional substance,
Formula (III) below:
ALB'(-F)-R'-T (III)
[In the formula,
A and L are the same as in formula (I) above;
R' and T are the same as in formula (II) above;
B' is a divalent group that includes a moiety generated by a reaction between a functional agent and a bioorthogonal functional group;
F is a functional substance. ] and (C) the affinity substance for the soluble protein, the cleavable moiety , cleaving a cleavable portion of a complex having a functional agent and a soluble protein or a salt thereof,
The following formula (V1):
L1-B'(-F)-R'-T (V1)
[In the formula,
L1 is (i′) a monovalent group containing a bioorthogonal functional group or (ii′) a monovalent group not containing a bioorthogonal functional group;
B', F, R' and T are the same as in formula (III) above. ], comprising producing a soluble protein having a functional substance or a salt thereof. A method for producing a soluble protein having a functional substance or a salt thereof,
(A) the following formula (II):
A-LB-R'-T (II)
[In the formula,
A is an affinity substance for soluble proteins;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B is (a) a divalent group that includes a bioorthogonal functional group or (a) a divalent group that does not include a bioorthogonal functional group;
R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein. ], cleaving the cleavable portion of a soluble protein having an affinity for soluble proteins and a soluble protein having a cleavable portion or a salt thereof,
Formula (IV) below:
L1-B-R'-T (IV)
[In the formula,
B, R' and T are the same as in formula (II) above;
L1 is (i') a monovalent group that contains a bioorthogonal functional group or (ii') a monovalent group that does not contain a bioorthogonal functional group. ], and (B) the soluble protein having a bioorthogonal functional group or a salt thereof represented by one or more functions by reacting with a substance,
The following formula (V2)
FL1'-B-R'-T (V2)
[In the formula,
B, R' and T are the same as in formula (IV) above;
L1' is a divalent group comprising a moiety produced by a reaction between a functional agent and (i') a monovalent group comprising a bioorthogonal functional group;
F is a functional substance. ], or the following formula (V3):
Fa-L1'-B'(-Fb)-R'-T (V3)
[In the formula,
R' and T are the same as in formula (IV) above;
L1' is the same as in formula (V2) above,
B' is a divalent group that includes a moiety generated by a reaction between a functional agent and a bioorthogonal functional group;
Fa and Fb are the same or different functional substances, respectively. ], comprising producing a soluble protein having a functional substance or a salt thereof. A method for producing a soluble protein having a functional substance or a salt thereof,
(A) the following formula (I):
A-L-B-R (I)
[In the formula,
A is an affinity substance for soluble proteins;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B is (a) a divalent group that includes a bioorthogonal functional group or (a) a divalent group that does not include a bioorthogonal functional group;
R is a reactive group for said soluble protein. ], the compound or its salt is reacted with a soluble protein,
Formula (II) below:
A-LB-R'-T (II)
[In the formula,
A, L, and B are the same as in formula (I) above R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein. ], and producing a soluble protein or a salt thereof having a cleavable moiety,
(B) cleaving a cleavable portion of a soluble protein having an affinity for said soluble protein and a soluble protein having a cleavable portion or a salt thereof,
Formula (IV) below:
L1-B-R'-T (IV)
[In the formula,
B, R' and T are the same as in formula (II) above;
L1 is (i') a monovalent group that contains a bioorthogonal functional group or (ii') a monovalent group that does not contain a bioorthogonal functional group. and (C) the soluble protein having a bioorthogonal functional group or a salt thereof represented by one or more functions by reacting with a substance,
The following formula (V2)
FL1'-B-R'-T (V2)
[In the formula,
B, R' and T are the same as in formula (IV) above;
L1' is a divalent group comprising a moiety produced by a reaction between a functional agent and (i') a monovalent group comprising a bioorthogonal functional group;
F is a functional substance. ], or the following formula (V3):
Fa-L1'-B'(-Fb)-R'-T (V3)
[In the formula,
R' and T are the same as in formula (IV) above;
L1' is the same as in formula (V2) above,
B' is a divalent group comprising a moiety generated by a reaction between a functional agent and a bioorthogonal functional group;
Fa and Fb are the same or different functional substances, respectively. ], comprising producing a soluble protein having a functional substance or a salt thereof. A soluble protein or a salt thereof regioselectively having a bioorthogonal functional group,
The soluble protein contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and the specific amino acid residue in a non-target region other than the target region 5 or more bioorthogonal functional groups with a regioselectivity of 30% or more for one or more specific amino acid residues contained in the target region via a linker that does not contain a peptide moiety A soluble protein or salt thereof having a regioselectively attached bioorthogonal functional group. Formula (IV) below:
L1-B-R'-T (IV)
[In the formula,
L1 is (i′) a monovalent group containing a bioorthogonal functional group or (ii′) a monovalent group not containing a bioorthogonal functional group;
B is (a) a divalent group that includes a bioorthogonal functional group or (b) a divalent group that does not include a bioorthogonal functional group;
R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein,
The soluble protein contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and the specific amino acid residue in a non-target region other than the target region 5 or more. ] and the structural unit represented by L1-BR' has a regioselectivity of 30% or more with respect to one or more specific amino acid residues contained in the target region of the soluble protein A soluble protein or salt thereof having a regioselectively attached bioorthogonal functional group. A method for producing a soluble protein or a salt thereof regioselectively having a bioorthogonal functional group, comprising:
Formula (II) below:
A-LB-R'-T (II)
[In the formula,
A is an affinity substance for soluble proteins;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B is (a) a divalent group that includes a bioorthogonal functional group or (a) a divalent group that does not include a bioorthogonal functional group;
R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein,
The soluble protein contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and the specific amino acid residue in a non-target region other than the target region 5 or more. ] and the structural unit represented by L1-BR' has a regioselectivity of 30% or more with respect to one or more specific amino acid residues contained in the target region of the soluble protein cleaving the bound affinity substance for the soluble protein and the cleavable portion of the soluble protein or salt thereof regioselectively carrying the cleavable portion,
Formula (IV) below:
L1-B-R'-T (IV)
[In the formula,
B, R' and T are the same as in formula (II) above;
L1 is (i') a monovalent group that contains a bioorthogonal functional group or (ii') a monovalent group that does not contain a bioorthogonal functional group. ] and the structural unit represented by L1-BR' has a regioselectivity of 30% or more with respect to one or more specific amino acid residues contained in the target region of the soluble protein A method comprising producing a soluble protein or salt thereof regioselectively having an attached bioorthogonal functional group. A soluble protein or a salt thereof regioselectively having a functional substance,
The soluble protein contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and the specific amino acid residue in a non-target region other than the target region 5 or more, and the functional substance binds to one or more specific amino acid residues contained in the target region with a position selectivity of 30% or more via a linker that does not contain a peptide portion. A soluble protein or a salt thereof that regioselectively has a functional substance. Formula (V) below:
F-(L1-B)'-R'-T (V)
[In the formula,
L1 is (i′) a monovalent group containing a bioorthogonal functional group or (ii′) a monovalent group not containing a bioorthogonal functional group;
B is (a) a divalent group that includes a bioorthogonal functional group or (b) a divalent group that does not include a bioorthogonal functional group;
The structural unit represented by (L1-B)' includes a moiety generated by a reaction between a functional substance and either one or both of the bioorthogonal functional groups in (i') and (a). is a divalent structural unit,
F is a functional substance,
R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein,
The soluble protein contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and the specific amino acid residue in a non-target region other than the target region 5 or more. ] and the structural unit represented by F-(L1-B)'-R' accounts for 30% or more of one or more specific amino acid residues contained in the target region of the soluble protein A soluble protein or a salt thereof regioselectively having a functional substance bound thereto with regioselectivity. A method for producing a soluble protein or a salt thereof regioselectively having a functional substance,
Formula (III) below:
ALB'(-F)-R'-T (III)
[In the formula,
A is an affinity substance for soluble proteins;
L is a cleavable linker that is a divalent group containing a cleavable moiety;
B' is a divalent group that includes a moiety generated by a reaction between a functional agent and a bioorthogonal functional group;
F is a functional substance,
R' is a moiety generated by reaction between a soluble protein and a reactive group;
T is a soluble protein,
The soluble protein contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and the specific amino acid residue in a non-target region other than the target region 5 or more. ] and the structural unit represented by ALB'(-F)-R' is 30 for one or more specific amino acid residues contained in the target region of the soluble protein. cleaving the cleavable portion of a complex or a salt thereof regioselectively having an affinity substance for a soluble protein, a cleavable portion, a functional substance and a soluble protein bound with a regioselectivity of ≥%, or Formula (IV) below:
L1-B-R'-T (IV)
[In the formula,
L1 is (i′) a monovalent group containing a bioorthogonal functional group or (ii′) a monovalent group not containing a bioorthogonal functional group;
B is (a) a divalent group that includes a bioorthogonal functional group or (b) a divalent group that does not include a bioorthogonal functional group;
R' is a moiety generated by reaction between a soluble protein and a reactive group;
T contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and 5 of the specific amino acid residues in a non-target region other than the target region It is a soluble protein containing more than ] and the structural unit represented by ALB'(-F)-R' is 30 for one or more specific amino acid residues contained in the target region of the soluble protein. A soluble protein or a salt thereof regioselectively having a bioorthogonal functional group bound with a regioselectivity of 10% or more is reacted with a functional substance,
Formula (V) below:
F-(L1-B)'-R'-T (V)
[In the formula,
L1 is (i′) a monovalent group containing a bioorthogonal functional group or (ii′) a monovalent group not containing a bioorthogonal functional group;
B is (a) a divalent group that includes a bioorthogonal functional group or (b) a divalent group that does not include a bioorthogonal functional group;
The structural unit represented by (L1-B)' includes a moiety generated by a reaction between a functional substance and either one or both of the bioorthogonal functional groups in (i') and (a). is a divalent structural unit,
F is a functional substance,
R' and T are the same as in formula (III) or (IV) above. ] and the structural unit represented by F-(L1-B)'-R' accounts for 30% or more of one or more specific amino acid residues contained in the target region of the soluble protein producing a soluble protein or a salt thereof regioselectively having a functional substance regioselectively attached to

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